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摘要植物次生代谢产物是临床药物的主要来源，也是新药开发的重要资源。然而，植物次生代谢产物由于含量稀少且结构复杂，难以进行化学合成，严重制约了其大规模生产。近年来，随着基因组测序技术以及生物探矿技术的发展，编码植物次生代谢产物的基因簇与合成途径不断涌现， 为复杂植物天然产物的合成生物学研究奠定了基础。植物基因簇往往长达数万至数十万个碱基对，传统的大片段基因组克隆方法依赖于繁琐耗时的基因组文库构建、筛选与拼接，难以满足植物基因簇的高通量精准克隆，因此迫切需要简单高效的克隆方法，将植物基因簇导入微生物进行异源表达以ᨀ高合成效率。为此，本研究建立了环化剪接技术(excisions by cyclization，EXCY)，首先利用 CRISPR/CRISPR-Cas9 系统将两个分别编码转录激活效应物与沉默抗性基因的EXCY 模块插入目的基因簇两端； 其次，筛选抗生素抗性突变体进行悬浮培养， 低温诱导EXCY模块上的FLP/lox系统将目的DNA片段剪接环化；最后，将所得质粒整合至酵母染色体进行基因编辑与异源表达。利用该方法，本文将 Papaver somniferum 中编码抗癌药物 Noscapine的基因簇（104-kb）克隆并导入酿酒酵母中，经内含子删除、启动子/终止子替换后，酵母工程菌株成功将Canadine底物转化为 Noscapine。EXCY 克隆在种间基因组上的剪接与整合功能为大片段基因簇克隆及编辑开辟了新途径。48050
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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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