Collaborative Research: Mechanisms for Cell Membrane Damage during Production of Biorenewable Fuels

合作研究：生物可再生燃料生产过程中细胞膜损伤的机制

• 批准号: 1604576
• 负责人: Jeffery Klauda
• 金额: $ 20万
• 依托单位: University of Maryland, College Park
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1604576&HistoricalAwards=false
• 关键词: Collaborative; Research; Mechanisms; Cell; Membrane

The conversion of plant biomass to biofuels by fermentation of biomass-derived sugars to alcohols is a sustainable route for renewable fuels production. However, during the fermentation process, the biofuel molecules produced are often toxic to the fermenting organism at high concentrations, which lowers the overall biofuel production capacity. One way to address this problem is redesign the membrane surrounding the fermenting cell, so that the organism can be more tolerant to high concentrations of biofuel dissolved in the liquid water surrounding it. This collaborative project will develop a fundamental understanding of how biofuel molecules such as ethanol interact with the cell membrane of yeast, a fermenting microorganism. The key innovation is use of sophisticated molecular dynamic simulation tools to model these interactions on computer. These studies will suggest strategies to target genetic engineering of the yeast cell to express cell membranes that improve the overall tolerance of the cell to high concentrations of biofuel, so that biofuel production is improved. The educational activities associated with this project include a middle- and high school outreach program designed to highlight how experiments and theory work together to solve important scientific problems, coordinated through programs at Iowa State University and the University of Maryland.The overall goal of this collaborative research is to gain a fundamental understanding of the cellular and biomolecular interactions of the microbial membranes with model biofuel molecules known to influence membrane disruption. The research will suggest how cell membranes can be engineered so that the cell is more tolerant to these biofuel molecules. The research plan will focus on how Saccharomyces cerevisiae membranes interact with model biofuel molecules or intermediates, including ethanol, octanoic acid, and n-butanol. These efforts will inform genetic engineering approaches to design cell membranes that improve the tolerance of the yeast cell to these biofuel molecules. Key membrane metrics include porosity, fluidity, hydrophobicity and rigidity. The research plan has three objectives. The first objective is to establish theoretical and experimental model membrane systems, using ethanol as a well-characterized model inhibitor. The theoretical approach will use molecular dynamics simulations to reveal how the molecular character and lipid composition of the membrane influence ethanol-mediated membrane disruption. The second objective is to probe the interaction of octanoic acid and n-butanol with model membrane systems developed under the first objective. More complex membrane systems will be considered to determine the effect of ergosterol, chain unsaturation, and lipid head groups on membrane disrupter toxicity. Membrane vesicles assembled in vitro will be compared vesicles made from whole cells as well as intact whole cells to establish model membrane of lipid mixtures to living systems. The third objective is to use molecular dynamics simulations to predict the improved tolerance of membranes with altered lipid head group concentrations, chain saturation, chain branching, and ergosterol concentration. Membranes with improved tolerance will be tested in vitro and then expressed and tested in engineered S. cerevisiae cells.

将植物生物质转化为生物燃料，通过发酵生物质衍生的糖转化为醇，是可再生燃料生产的可持续途径。然而，在发酵过程中，高浓度产生的生物燃料分子往往对发酵生物有毒，从而降低了生物燃料的整体生产能力。解决这个问题的一种方法是重新设计发酵细胞周围的膜，这样生物就能更耐受溶解在周围液态水中的高浓度生物燃料。这个合作项目将对生物燃料分子（如乙醇）如何与酵母（一种发酵微生物）的细胞膜相互作用有一个基本的了解。关键的创新是使用复杂的分子动力学模拟工具在计算机上模拟这些相互作用。这些研究将提出针对酵母细胞的基因工程的策略，以表达提高细胞对高浓度生物燃料的总体耐受性的细胞膜，从而提高生物燃料的产量。与此项目相关的教育活动包括一个初高中推广项目，旨在强调实验和理论如何结合起来解决重要的科学问题，并通过爱荷华州立大学和马里兰大学的项目进行协调。这项合作研究的总体目标是对微生物膜与已知影响膜破坏的模型生物燃料分子之间的细胞和生物分子相互作用有一个基本的了解。这项研究将建议如何改造细胞膜，使细胞对这些生物燃料分子更具耐受性。该研究计划将重点关注酿酒酵母膜如何与生物燃料模型分子或中间体（包括乙醇、辛酸和正丁醇）相互作用。这些努力将为基因工程方法设计细胞膜提供信息，以提高酵母细胞对这些生物燃料分子的耐受性。关键的膜指标包括孔隙度、流动性、疏水性和刚性。研究计划有三个目标。第一个目标是建立理论和实验模型膜系统，使用乙醇作为表征良好的模型抑制剂。理论方法将使用分子动力学模拟来揭示膜的分子特性和脂质组成如何影响乙醇介导的膜破坏。第二个目标是探索辛酸和正丁醇与第一个目标下开发的模型膜系统的相互作用。将考虑更复杂的膜系统来确定麦角甾醇、链不饱和和脂质头基团对膜干扰物毒性的影响。将体外组装的膜囊泡与完整的全细胞组装的膜囊泡进行比较，建立脂质混合物到活体系统的模型膜。第三个目标是使用分子动力学模拟来预测随着脂质头基团浓度、链饱和度、链分支和麦角甾醇浓度的改变，膜的耐受性得到改善。耐受性提高的膜将在体外测试，然后在工程酿酒葡萄球菌细胞中表达和测试。