Discovering the xylan-sensing pathway in the filamentous fungus Neurospora crassa

发现丝状真菌粗糙脉孢菌中的木聚糖传感途径

• 批准号: 9039464
• 负责人: Lori B Huberman
• 金额: $ 5.61万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9039464
• 关键词: Animals; Biological; Carbohydrates; Carbon; Cell Wall; Cells; Cellular biology; Cleaved cell; Complex; Diabetes Mellitus; Directed Molecular Evolution; Disaccharides; Disease; Drug resistance; Enzymes; Eukaryota; Genes; Genetic; Genetic Transcription; Glucose; Goals; Human; Infection; Learning; Light; Malignant Neoplasms; Mammals; Maps; Metabolic; Methods; Microbe; Modeling; Molds; Molecular; Monosaccharides; Mutate; Mutation; Neurospora crassa; Nutrient; Obesity; Organism; Pathway interactions; Plant Components; Plants; Polysaccharides; Production; Proteins; Regulation; Regulon; Resources; Role; Saccharomyces cerevisiae; Saccharomycetales; Scanning; Scheme; Signal Pathway; Signal Transduction; Source; Sucrose; Techniques; Time; Transcriptional Regulation; Transgenic Organisms; Work; Xylans; Yeast Model System; Yeasts; cell growth; design; detection of nutrient; drug sensitivity; fungus; glucose uptake; hemicellulase; improved; interest; mutant; promoter; public health relevance; response; screening; sugar; synthetic biology; tool; transcription factor; tumor progression

 DESCRIPTION (provided by applicant): Identifying and utilizing the nutrients available in the most efficient manner is a challenge common to all organisms. In humans inaccurate or failed nutrient sensing can result in a variety of diseases including diabetes and obesity, and cancer progression has been shown to rely on increased glucose uptake and changes in nutrient sensing. Many of the nutrient sensing pathways are conserved from yeast to humans, and studies on nutrient sensing in unicellular eukaryotes have been instrumental in elucidating nutrient sensing pathways in humans. However, much of the work on nutrient signaling in unicellular eukaryotes has been done in budding yeast, which has a fairly limited carbohydrate utilization repertoire. Unlike budding yeast, the model filamentous fungus, Neurospora crassa, is capable of utilizing a wide variety of carbohydrates: from simple sugars to the complex sugar chains found in plant cell walls. In order to efficiently exploit the available resources, N. crass must be capable of sensing and responding to the presence of these different carbohydrates. Several transcription factors have been identified in N. crassa that activate the transcription of plant cell wall-degrading enzymes. One of these is XLR1, which activates the transcription of hemicellulases when in the presence of the plant cell wall component xylan. However, while it seems evident that XLR1 must be activated in order to induce expression of hemicellulases, the method by which this is achieved is still unclear. The goal of this project is to identify and characterize upstream regulators of XLR1 and their interactions with other nutrient sensing pathways in N. crassa, which will be accomplished through the completion of the following three specific aims. The first is to screen for mutants in which XLR1 activation is either constitutive o uninducible to identify the genes involved in the xylan-sensing pathway and characterize their function and role in xylan sensing. The second aim is to use directed evolution to probe more subtle genetic interactions both within the xylan-sensing pathway and between the xylan-sensing pathway and other nutrient sensing pathways to identify mutations which optimize N. crassa for rapid and accurate xylan sensing and utilization as well as to understand how the xylan-sensing pathway fits into the larger scheme of pathways that assess the metabolic state of the cell. And the third aim is to use synthetic biology to reconstruct the xylan-sensing pathway in the budding yeast, Saccharomyces cerevisiae, which is not able to use xylan as a carbon source, using the optimized genes identified in the first two aims to validate the identification o genes involved in the xylan-sensing pathway. The completion of these aims should shed light on the xylan-sensing pathway in N. crassa and its interaction with other cellular signaling pathways. We expect this to improve our overall understanding of cellular signaling throughout the eukaryotic realm , since scanning the diversity of molecular mechanisms involved in signaling pathways is helpful in elucidating broad biological paradigms.

 描述（由申请人提供）：以最有效的方式识别和利用可用的营养素是所有生物体共同面临的挑战。在人类中，不准确或失败的营养感测可导致多种疾病，包括糖尿病和肥胖症，并且癌症进展已被证明依赖于增加的葡萄糖摄取和营养感测的变化。从酵母到人类，许多营养传感途径都是保守的，单细胞真核生物中营养传感的研究有助于阐明人类的营养传感途径。然而，在单细胞真核生物中营养信号的大部分工作已经在芽殖酵母中完成，芽殖酵母具有相当有限的碳水化合物利用库。与芽殖酵母不同，模式丝状真菌粗糙脉孢菌能够利用各种碳水化合物：从简单的糖到植物细胞壁中发现的复杂糖链。为了有效地利用现有资源，N。crass必须能够感知和响应这些不同碳水化合物的存在。在N. crassa，其激活植物细胞壁降解酶的转录。其中之一是XLR 1，其在植物细胞壁组分木聚糖存在时激活半纤维素酶的转录。然而，虽然似乎很明显，XLR 1必须被激活以诱导半纤维素酶的表达，但实现这一点的方法仍不清楚。本项目的目标是确定和表征XLR1的上游调控因子及其与N.这将通过完成以下三个具体目标来实现。首先是筛选XLR1激活是组成型或不可诱导的突变体，以鉴定参与木聚糖传感途径的基因，并表征其在木聚糖传感中的功能和作用。第二个目标是使用定向进化来探测木聚糖传感途径内以及木聚糖传感途径与其他营养传感途径之间的更微妙的遗传相互作用，以识别优化N的突变。crassa用于快速和准确的木聚糖传感和利用，以及了解木聚糖传感途径如何适应评估细胞代谢状态的更大途径方案。第三个目标是利用合成生物学技术重建木聚糖传感通路 在不能使用木聚糖作为碳源的芽殖酵母酿酒酵母中，使用前两者中鉴定的优化基因旨在验证参与木聚糖传感途径的基因的鉴定。这些目标的实现将为N. crassa及其与其他细胞信号通路的相互作用。我们希望这能提高我们对整个真核领域细胞信号传导的整体理解，因为扫描信号通路中涉及的分子机制的多样性有助于阐明广泛的生物学范式。