DNA Assembly via Reiterative Recombination

描述（由申请人提供）：代谢工程已成为生产具有重要治疗意义的天然产物及其类似物的一种有前途的策略。然而，生物合成基因通路的构建和操作是制造复杂天然产物及其衍生物的细胞工程努力的关键瓶颈，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一种有效的多基因DNA结构体内组装策略。在这里，我们建议通过优化酵母中萜类化合物（特别是紫杉醇中间体）的产量来挑战我们的技术，以开发代谢工程应用。如果成功，这项研究将

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.