CAREER: Glucan phosphatases: a key to designer starches and plant energy storage

智力优势：作为植物和藻类的主要能量储存，淀粉是人类和动物食物的核心成分，也是许多制造过程的关键成分。此外，淀粉既是第一代生物燃料，也是未来微藻氢气和石油生产的关键。因此，有必要阐明控制淀粉代谢的途径，以便开发新的策略来操纵它们并满足日益增长的淀粉需求。调节淀粉代谢的一个关键途径——也是淀粉降解所必需的——是淀粉外葡聚糖中葡萄糖残基的可逆磷酸化，使葡聚糖水解酶能够进入颗粒表面。当两个葡聚糖二激酶通过磷酸化葡萄糖单位使淀粉外葡聚糖链溶解时，这个连续的循环开始。淀粉磷酸化使淀粉酶结合淀粉表面，并以葡萄糖和麦芽糖的形式释放储存的能量；然而，淀粉酶不会经过磷酸基团。两种葡聚糖磷酸酶通过去除磷酸基团并允许葡聚糖水解来重置循环。缺乏葡聚糖磷酸酶的植物表现为淀粉过量，生长受损，淀粉磷酸化增加，淀粉分解中间体积累增加。淀粉可逆性磷酸化的生物学研究取得了一定进展，但调控葡聚糖磷酸酶功能的分子机制尚不清楚。该项目通过采用各种生化和生物物理技术解决了这一重要途径的关键信息缺口。本研究将定义葡聚糖磷酸酶的功能、动力学、结构和调控，以及工程葡聚糖磷酸酶的生成和评价。这项工作的完成将明确葡聚糖磷酸酶在淀粉修饰和降解中的作用，为这些酶的生物技术开发提供必要的见解。更广泛的影响：不断增长的需求导致了淀粉在食品、生物燃料和工业制造商之间的竞争。另一个令人担忧的问题是，淀粉加工过程中使用危险化学品对其进行改性，以供工业应用。因此，需要创新的策略来增加淀粉产量，并使用危害较小的方法来改变淀粉的生物物理特性。该项目包含综合和协同的职业目标，将影响提高淀粉产量和产生设计淀粉的能力。这个项目涉及几个层次的多学科培训。1)研究生将接受国内（肯塔基州和加利福尼亚州）和国际（瑞士）的实验室技能、科学伦理、蛋白质纯化、酶动力学、晶体学、DXMS方法以及数据分析和表达方面的培训。此外，他们将在职业选择方面得到指导。2)本科生，包括NSF REU学生和来自阿巴拉契亚地区的学生，将接受基本的实验室技能和实验室使用技术的培训。他们还将获得职业选择方面的指导。3)将淀粉代谢的研究融入到高水平的本科课程中，加强正规的本科课程。4)在州政府的活动中，将向高中生和本科生以及公众展示食品生产、生物燃料和全球变暖等社会和环境问题基础研究的重要性。此外，研究结果将发表在同行评议的期刊上，并在地方、区域、国家和国际科学跨学科会议上发表。

Intellectual Merit: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.