Yeast mitochondrial engineering: A new technology for synthetic biology and metab

酵母线粒体工程：合成生物学和代谢新技术

• 批准号: 8316782
• 负责人: Jose L Avalos
• 金额: $ 5.94万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8316782
• 关键词: 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); Acetone; Affect; Affinity; Alcohols; Antibodies; Biological Products; Biology; Biosensor; Branched-Chain Amino Acids; Butanols; C-terminal; Catabolism; Cells; Chemicals; Chemistry; Cloning; Clostridium; Complex; Cytoplasm; Development; Dimensions; Drug Industry; Elements; Engineering; Enzymes; Ethanol; Expression Library; Food; Foundations; Gene Deletion; Gene Expression; Gene Mutation; Generations; Genes; Genetic; Goals; Health; Health Benefit; Hormones; Human; Industry; Initiator Codon; Isoenzymes; Left; Metabolic; Metabolic Pathway; Metabolism; Methods; Mitochondria; Modification; Molecular Biology; Molecular Cloning; Organelles; Organism; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Physiology; Plasmids; Production; Proteins; Reporter; Research; Resources; Saccharomyces cerevisiae; Sampling; Screening procedure; Series; Signal Transduction; Site; Speed; Standardization; Terminator Codon; Vaccines; Yeasts; base; cost; design; drug production; gene cloning; genetic manipulation; high throughput screening; improved; interest; microorganism; mutant; new technology; overexpression; promoter; protein expression; success; synthetic biology; tool; vector

DESCRIPTION (provided by applicant): Microorganisms are widely used in the pharmaceutical industry to make products that are invaluable to human health such as drugs, their precursors, and biological agents such as vaccines, antibodies and hormones. Yeast is the organism of choice for these important applications due to the ease of its genetic manipulation, deep understanding of its basic biology, and the significant industrial advantages it has over other microorganisms. However, metabolic engineering efforts in yeast have focused mostly on the overexpression of enzymes in the cytoplasm, leaving the wealth of resources in its mitochondria essentially untapped. The goal of this project is to develop the tools, methods and strategies to engineer mitochondria in the yeast Saccharomysces cerevisiae, and gain access to the many benefits provided by this organelle for the production of new compounds important to the pharmaceutical and other industries. The first aim is to develop a new standardized vector series for protein expression in yeast (including targeted expression to mitochondria) to expedite and reduce the costs of cloning, assembly, troubleshooting and optimization of engineered pathways. This new vector series will help overcome many aspects of molecular biology that are often bottlenecks in any metabolic engineering project. The second aim is to demonstrate the benefits of mitochondrial engineering in the construction of heavy alcohol biosynthetic pathways, which stand to gain much by this new technology. This will involve the screening of pathway components, their assembly, and implementation using the new vector series described above. The third aim is to identify genes, mutations or conditions that affect mitochondrial physiology in favor of the engineered heavy alcohol biosynthetic pathways. This will entail the development of a new metabolic flux biosensor, which will allow the design of high throughput assays to screen thousands of strains rather than the few dozens that are feasible with current methods. The broad, long-term implication of this project is in laying the foundation for mitochondrial engineering, a new technology applicable to synthetic biology and metabolic engineering for countless benefits to health research and the pharmaceutical industry. PUBLIC HEALTH RELEVANCE: Baker's yeast, Saccharomyces cerevisiae, is a preferred microorganism for applications relevant to the pharmaceutical and other industries, including for the production of drugs and other biological agents such as vaccines. While the ease to carry out the genetic modifications required for these applications is one of the attributes that has made yeast such a useful organism, the wealth of resources provided by its mitochondria (the cell's powerhouse) remain essentially untapped for similar applications. This project focuses on the development of mitochondrial engineering to harness the unique advantages of this organelle, which will enable new yeast applications in synthetic biology and metabolic engineering to benefit health research and the pharmaceutical industry.

描述(申请人提供)：微生物在制药工业中被广泛用于制造对人类健康有无价价值的产品，如药物、其前体，以及生物制剂，如疫苗、抗体和激素。酵母是这些重要应用的首选有机体，因为它的基因操作容易，对其基本生物学有深入的了解，以及它相对于其他微生物具有显著的工业优势。然而，酵母中的代谢工程努力主要集中在细胞质中酶的过度表达上，留下了线粒体中丰富的资源基本上没有被开发。该项目的目标是开发工具、方法和策略来在酿酒酵母中改造线粒体，并获得该细胞器为生产对制药和其他行业重要的新化合物所提供的许多好处。第一个目标是开发一种新的标准化载体系列，用于在酵母中表达蛋白质(包括定向表达到线粒体)，以加快并降低工程途径的克隆、组装、故障排除和优化的成本。这一新的载体系列将有助于克服分子生物学的许多方面，这些方面往往是任何代谢工程项目的瓶颈。第二个目标是证明线粒体工程在构建重醇生物合成途径方面的好处，这项新技术将使其获益良多。这将涉及使用上述新载体系列筛选途径组件、组装和实施。第三个目标是识别影响线粒体生理学的基因、突变或条件，以支持工程重醇生物合成途径。这将需要开发一种新的代谢通量生物传感器，它将允许设计高通量分析来筛选数千个菌株，而不是目前方法可行的几十个菌株。该项目的广泛和长期影响是为线粒体工程奠定基础，线粒体工程是一种适用于合成生物学和代谢工程的新技术，可以为健康研究和制药行业带来无数好处。 与公共健康相关：面包酵母，酿酒酵母，是与制药和其他行业相关的应用的首选微生物，包括生产药物和其他生物制剂，如疫苗。虽然易于进行这些应用所需的基因修改是使酵母成为如此有用的有机体的属性之一，但其线粒体(细胞的动力源)提供的丰富资源基本上仍未被用于类似的应用。该项目专注于线粒体工程的开发，以利用这种细胞器的独特优势，使酵母在合成生物学和代谢工程中的新应用能够造福于健康研究和制药工业。