DNA Assembly via Reiterative Recombination

通过重复重组进行 DNA 组装

基本信息

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
项目类别：
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

项目摘要

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具有治疗重要的天然产品及其类似物的有希望的策略。然而，生物合成基因途径的构建和操纵为努力设计复杂天然产物及其衍生物的努力构成了关键的瓶颈，并且需要新一代的变革性技术来克服这一障碍。为此，我们已经开发了重组，作为多基因DNA构建体体内组装有效组装的策略。在这里，我们建议通过在优化型萜类产量的背景下对代谢工程应用开发代谢工程应用的技术，特别是针对酵母中紫杉醇中间体的技术。如果成功，这项研究将将天然产品生物合成途径的库的构造成为一个常规，负担得起且高效的过程，而非专家可以在没有任何专业设备的情况下进行。公共卫生相关性：该提案的目的是为多基因DNA构建体的体内组装创建一种变革性技术。该技术将极大地促进用于代谢工程和其他合成生物学应用的工程菌株的构建。通过为组装大型DNA构建体提供强大，高效且可访问的方法，该技术（如果成功）将大大努力生产重组微生物中重要的天然产物。