Engineering Molecular Transport Proteins for Improved Xylose Uptake in Yeast

Alper/1067506项目总结分子转运蛋白本质上限制了细胞生物加工的最大速率，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，而专注于细胞内途径酶。 在外源糖利用的情况下，转运速率通常可以成为该过程的主要速率限制步骤。 具体地，酿酒酵母中的木糖转运受到缺乏具有（a）高流入速率和（B）低葡萄糖抑制的特异性转运蛋白的限制。 尽管该领域的先前工作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，采用双性状优化木糖转运蛋白，以证明工程分子转运蛋白的效用。 我们的团队将采用定向进化的三个确定的木糖转运蛋白使用开发的木糖生物传感器作为筛选改进的突变体。 我们小组正在进行的初步工作支持这一做法。 我们将研究搜索轨迹对选择具有双性状改善的转运蛋白（即增加木糖转运速率和降低葡萄糖抑制）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质的功能。 这种分子转运蛋白的蛋白质工程与传统途径工程的新组合为细胞和代谢工程提供了一种变革性和强大的方法。 这种方法的新奇在于通过定向进化方法对效率和糖选择性的双重性状进行改进运输速率的方法的检查。智力优点这项研究测试了基本假设，即（1）木糖运输速率和葡萄糖抑制是由关键氨基酸残基或结构域控制的，这些残基或结构域可以通过诱变鉴定，以及（2）提高木糖转运蛋白的净转运速率和葡萄糖抑制水平将导致酵母细胞具有提高的木糖利用率。 在此过程中，本研究将确定在分子转运蛋白上采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这些蛋白中哪些残基负责结合亲和力、抑制和速率的更详细的理解。 关键残基和功能表征的解剖将量化这些工程转运蛋白提供的变化幅度。 在这里评估的转运效率和选择性之间的连接的理解将提供新的洞察转运蛋白的潜力和进化的优势和成本酵母拥有广泛的或特定的糖转运蛋白。 因此，这种方法推进了对代谢工程的理解，并将其应用于改善产品通量。更广泛的影响进化或工程化细胞中转运蛋白功能的能力将导致更高效和专业的生物技术过程。 具体对于该项目，鉴定改进的木糖转运蛋白（具有增加的木糖转运速率和降低的葡萄糖抑制）将对木质纤维素生物质转化具有很大的工业用途。 在这方面，这种方法增加了一个新的维度，以改善代谢通量和工程细胞的生化生产。 这项研究将支持一个研究生和几个本科生研究人员的跨学科研究。 本科生将积极从新生研究计划（FRI）中招募，这是一个NSF支持的项目，旨在改善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学教师Michelle Halvorsen女士合作，更广泛地推广到K-12社区。 在夏天，哈尔沃森夫人，沿着从学校的学生，将在实验室进行研究，并开发实验室模块带回高中课堂。 这一举措将有助于将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普通科学领域的领导者。

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.