DNA Assembly via Reiterative Recombination

通过重复重组进行 DNA 组装

• 批准号: 8258568
• 负责人: VIRGINIA W CORNISH
• 金额: $ 28.04万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8258568
• 关键词: Adoption; Affect; Anabolism; Biological Factors; Carbon; Cell Culture Techniques; Cells; Chromosomes; Codon Nucleotides; Commit; Communities; Complementary DNA; Complex; Cytochrome P450; DNA; Development; Double Strand Break Repair; Engineering; Enzymes; Equipment; Generations; Genes; Genetic; Genetic Recombination; Goals; In Vitro; Individual; Laboratories; Length; Libraries; Metabolic; Metabolic Pathway; Methods; Modeling; Molecular Biology Techniques; Organism; Paclitaxel; Pathway interactions; Pigments; Plants; Process; Production; Recombinants; Reporter Genes; Research; Resources; Skeleton; Solutions; Technology; Terpenes; Terpenoid Biosynthesis Pathway; Testing; Therapeutic; Time; Transplantation; Work; Yeasts; analog; base; cellular engineering; combinatorial; design; directed evolution; empowered; feeding; homologous recombination; improved; in vivo; isopentenyl pyrophosphate; lycopene; microbial host; microorganism; overexpression; promoter; synthetic biology; taxadiene

DESCRIPTION (provided by applicant): Metabolic engineering has emerged as a promising strategy for the production of therapeutically important natural products and their analogs. However, the construction and manipulation of pathways of biosynthetic genes poses a key bottleneck to efforts to engineer cells for the production of complex natural products and their derivatives, and a new generation of transformative technologies will be needed to overcome this barrier. Towards this end, we have developed Reiterative Recombination as a strategy for the efficient in vivo assembly of multi-gene DNA constructs. Here we propose to develop our technology for metabolic engineering applications by challenging it in the context of optimizing terpenoid yields, specifically for taxol intermediates, in yeast. If successful, this research will make the construction of libraries of natural product biosynthetic pathways a routine, affordable, and highly efficient process that can be carried out by the non-expert without any specialized equipment. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to create a transformative technology for in vivo assembly of multi-gene DNA constructs. This technology will greatly facilitate the construction of engineered strains for metabolic engineering and other synthetic biology applications. By providing a robust, efficient, and accessible method for the assembly of large DNA constructs, this technology, if successful, will significantly further efforts to produce therapeutically important natural products in recombinant microorganisms.

描述（由申请人提供）：代谢工程已成为生产治疗上重要的天然产物及其类似物的有前景的策略。然而，生物合成基因途径的构建和操作对改造细胞以生产复杂天然产物及其衍生物的努力构成了关键瓶颈，需要新一代的转化技术来克服这一障碍。为此，我们开发了重复重组作为多基因 DNA 构建体体内高效组装的策略。在这里，我们建议通过在酵母中优化萜类化合物产量（特别是紫杉醇中间体）的背景下挑战它来开发我们的代谢工程应用技术。如果成功的话，这项研究将 使天然产物生物合成途径文库的构建成为一种常规、负担得起且高效的过程，可由非专家无需任何专门设备即可进行。 公共健康相关性：该提案的目的是创建一种用于多基因 DNA 构建体体内组装的变革性技术。该技术将极大地促进代谢工程和其他合成生物学应用的工程菌株的构建。通过为大型 DNA 构建体的组装提供稳健、高效且易于使用的方法，该技术如果成功，将显着进一步努力生产 重组微生物中具有治疗作用的重要天然产物。