Engineering Molecular Transport Proteins for Improved Xylose Uptake in Yeast

工程分子运输蛋白以改善酵母中的木糖吸收

• 批准号: 1067506
• 负责人: Hal Alper
• 金额: $ 36.07万
• 依托单位: University of Texas at Austin
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1067506&HistoricalAwards=false
• 关键词: Engineering; Molecular; Transport; Proteins; Improved

Alper/ 1067506Project Summary Molecular transporters inherently limit the maximal rate of cellular bioprocessing since they control the first main step of any metabolic pathway. Yet, traditional pathway engineering approaches often overlook this aspect of cellular function and focus on intracellular pathway enzymes instead. In the case of exogenous sugar utilization, transport rate can often become the predominant rate limiting step of the process. Specifically, xylose transport in Saccharomyces cerevisiae is limited by the lack of a specific transporter possessing (a) high influx rates and (b) low glucose inhibition. These two limitations remain despite prior work in the field focused on heterologous expression of transporters or global evolutionary approaches. Our research team proposes a novel approach that employs dual-trait optimization of xylose transporters to demonstrate the utility of engineering molecular transport proteins. Our team will employ directed evolution on three identified xylose transporters using a developed xylose biosensor as a screen for improved mutants. This approach is supported by ongoing preliminary work by our group. We will examine the importance of search trajectory on selecting for transporters with dual-trait improvement (namely increased xylose transport rate and decreased glucose inhibition). Finally, select mutant transporters will be evaluated to understand their biochemical and genetic basis, providing insight into the function of these proteins. This novel combination of protein engineering of molecular transporters with traditional pathway engineering provides a transformative and powerful approach to cellular and metabolic engineering. The novelty of this approach resides in the examination of methods for improving transport rates via a directed evolution approach for the dual traits of efficiency and sugar selectivity.Intellectual Merit This research tests the basic hypotheses that (1) xylose transport rates and glucose inhibition are controlled by key amino acid residues or structural domains that may be identified through mutagenesis and (2) improving the net transport rate and glucose inhibition level of xylose transporters will lead to yeast cells with improved xylose utilization rates. In doing so, this research will determine the best strategies and search trajectories for employing protein engineering on molecular transporters in order to optimize dual traits. The genetic, biochemical, and functional analysis of mutant transporter proteins can uncover a more detailed understanding of which residues in these proteins are responsible for binding affinity, inhibition, and rates. A dissection of critical residues and functional characterization will quantify the magnitude of change provided by these engineered transporters. The understanding of the connection between transporter efficiency and selectivity evaluated here will provide novel insight into transporter potential and evolutionary advantages and costs for yeast to possess either broad or specific sugar transporters. Therefore, this approach advances the understanding of metabolic engineering and its application to improving product flux.Broader Impacts The ability to evolve or engineer transporter function in cells will lead to more efficient and specialized biotechnological processes. Specific for this project, the identification of improved xylose transporter proteins (with increased xylose transport rates and decreased glucose inhibition) would be of great industrial use for lignocellulosic biomass conversion. In this regard, this approach adds a novel dimension towards improving metabolic flux and engineering cells for biochemical production. This research will support the interdisciplinary study of one graduate student and several undergraduate researchers. Undergraduate students will be actively recruited from the Freshmen Research Initiative (FRI), an NSF supported program to improve under-represented minority participation in science research. In addition, this work will allow for a broader outreach to the K-12 community through collaboration with Mrs. Michelle Halvorsen, a biology teacher at the Texas School for the Deaf. During the summer, Mrs. Halvorsen, along with a student from the school, will conduct research in the lab and develop laboratory modules to bring back to the high school classroom. This initiative will help bring science outreach to the deaf community, a population often overlooked in STEM initiatives. The resulting broad, interdisciplinary training will allow students and researchers to become leaders in the field of metabolic and cellular engineering and in the general sciences.

分子转运蛋白固有地限制了细胞生物处理的最大速率，因为它们控制着任何代谢途径的第一个主要步骤。然而，传统的途径工程方法往往忽略了细胞功能的这一方面，而将重点放在细胞内途径酶上。在外源糖利用的情况下，运输速率通常可以成为该过程的主要速率限制步骤。具体来说，木糖在酿酒酵母中的运输受到缺乏具有(a)高内流率和(b)低葡萄糖抑制的特定转运体的限制。尽管先前的研究主要集中在转运蛋白的异源表达或全局进化方法上，但这两个限制仍然存在。我们的研究小组提出了一种新的方法，利用木糖转运蛋白的双性状优化来证明工程分子转运蛋白的实用性。我们的团队将使用开发的木糖生物传感器作为改进突变体的筛选，对三种已确定的木糖转运蛋白进行定向进化。这种方法得到了我们小组正在进行的初步工作的支持。我们将研究搜索轨迹在选择具有双性状改善的转运体（即木糖转运率增加和葡萄糖抑制降低）中的重要性。最后，将对选择的突变转运蛋白进行评估，以了解其生化和遗传基础，从而深入了解这些蛋白的功能。这种分子转运蛋白工程与传统途径工程的新结合为细胞和代谢工程提供了一种变革性和强有力的方法。这种方法的新颖之处在于通过定向进化方法来提高效率和糖选择性的双重特征的运输率。本研究验证了以下基本假设：(1)木糖转运率和葡萄糖抑制是由关键氨基酸残基或结构域控制的，这些氨基酸残基或结构域可以通过诱变识别；(2)提高木糖转运体的净转运率和葡萄糖抑制水平将导致酵母细胞提高木糖利用率。在此过程中，本研究将确定在分子转运体上使用蛋白质工程的最佳策略和搜索轨迹，以优化双重性状。突变转运蛋白的遗传、生化和功能分析可以揭示更详细的了解这些蛋白中的哪些残基负责结合亲和力、抑制和速率。关键残基的解剖和功能表征将量化这些工程转运体提供的变化幅度。对转运体效率和选择性之间的联系的理解将为酵母拥有广泛或特定的糖转运体的转运体潜力和进化优势和成本提供新的见解。因此，该方法促进了对代谢工程的理解及其在提高产品通量方面的应用。更广泛的影响进化或设计细胞中转运蛋白功能的能力将导致更有效和专门的生物技术过程。具体到这个项目，鉴定改进的木糖转运蛋白（增加木糖运输速率和降低葡萄糖抑制）将在木质纤维素生物质转化方面具有重要的工业用途。在这方面，这种方法为改善代谢通量和生物化学生产的工程细胞增加了一个新的维度。这项研究将支持一名研究生和几名本科生的跨学科研究。本科生将从新生研究计划（FRI）中积极招募，FRI是美国国家科学基金会支持的一项计划，旨在改善未被充分代表的少数民族参与科学研究的情况。此外，这项工作将通过与德克萨斯州聋人学校的生物老师米歇尔·哈尔沃森夫人的合作，扩大到K-12社区。在暑假期间，哈尔沃森女士将与学校的一名学生一起在实验室进行研究，并开发实验室模块，并将其带回高中课堂。这项倡议将有助于把科学推广到聋人社区，这是一个在STEM倡议中经常被忽视的群体。由此产生的广泛的跨学科培训将使学生和研究人员成为代谢和细胞工程领域以及一般科学领域的领导者。