基于环化剪接的植物基因簇克隆与重构
毕业论文关键词：植物次生代谢、基因簇、克隆、EXCY、Noscapine
Title Excisions by Cyclization for Targeted Cloning and Refactoring of Plant Gene Clusters
Abstract Plant secondary metabolites represent a large class of natural products of high pharmaceutical value, which makes them a great source of new drugs in pharmaceutical industry. However, large-scale production of natural products-based pharmaceuticals is limited, due to low abundance in planta and the complex structures. With the rapid developments of genomics and biosynthesis, the genes and biosynthetic pathways of many plant secondary metabolites recently have been elucidated, providing a new access to the biosynthesis of complex natural compounds. The rise of gene clusters in plants calls for simple and efficient method for cloning and refactoring of the gene clusters for heterologous expression. While owing to the scarcity of efficient procedures to connect plant gene to molecules, few gene clusters from plants have been investigated to date. To address these issues, we developed a technology termed excisions by cyclization (EXCY). Before cyclization, the two EXCY cassettes that each contains a transcription activator-like effector and a silent antibiotic resistance gene were simultaneously integrated into the flanking regions of the target sequence by means of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, and only the double insertion mutants survive after antibiotic selection. When cyclized by cooling, the plasmid formed that contains the target gene cluster was extracted and transformed into yeast for refactoring, thus streamlining the heterologous expression of complex metabolic pathway. With this method, we directly transferred the 104-kb gene cluster that was responsible for the biosynthesis of Noscapine from Papaver somniferum into Saccharomyces cerevisiae. After intron deletion and promoter/terminator substitution, the engineered yeast successfully transformed canadine into Noscapine. Finally, the ability to integrate and excise from genome using EXCY opens up engineering modalities for large genomic capture and refactoring.
Keywords: plant secondary metabolites, gene cluster, cloning, EXCY, Noscapine
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