Activation of Naringenin and Kaempferol through Pathway Refactoring in the Endophyte Phomopsis
Activation of Naringenin and Kaempferol through Pathway Refactoring in the Endophyte Phomopsis
复制标题
在内生拟茎点霉中通过途径重构激活柚皮素和山奈酚
DOI：
--
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Chuan-Chao Dai
中科院分区：
文献类型：
--
作者：
Mei Wu;Da-Chun Gong;Qian Yang;Meng-Qian Zhang;Yan-Zhen Mei;Chuan-Chao Dai
在植物内生真菌枫香拟茎点霉（Phomopsis liquidambaris）中发现了丰富的天然产物基因簇，但大多数基因簇是沉默的。在此，即插即用的DNA组装工具已被应用于枫香中的类黄酮合成。以S. cerevisiae，E. coli和P. liquidambaris分别与筛选标记URA、HYGR和HYGR进行筛选。每个片段或盒通过重叠延伸PCR与S中至少40 - 50 bp的同源臂连续组装。酿酒酵母用于产生新载体。根据mCherry报告基因的荧光强度，通过DNA拼接技术筛选出7个天然启动子，其中2个为新启动子。查尔酮合成酶是该途径的关键酶。采用补料分批发酵的方法对柚苷元和山奈酚途径进行了改造和激活，柚苷元和山奈酚的效价分别达到121.53 mg/L和75.38 mg/L。本研究将为次生代谢产物的生物合成提供有效的指导。
Abundant gene clusters of natural products are observed in the endophytic fungus Phomopsis liquidambaris; however,most of them are silent. Herein, a plug-and-play DNA assembly tool has been applied for flavonoid synthesis in P. liquidambaris. A shuttle plasmid was constructed based on S. cerevisiae, E. coli, and P. liquidambaris with screening markers URA, Amp, and hygR,respectively. Each fragment or cassette was successively assembled by overlap extension PCR with at least 40−50 bp homologous arms in S. cerevisiae for generating a new vector. Seven native promoters were screened by the DNA assembly based on the fluorescence intensity of the mCherry reporter gene in P. liquidambaris, and two of them were new promoters. The key enzyme chalcone synthase was the limiting step of the pathway. The naringenin and kaempferol pathways were refactored and activated with.the titers of naringenin and kaempferol of 121.53 mg/L and 75.38 mg/L in P. liquidambaris using fed-batch fermentation, respectively. This study will be efficient and helpful for the biosynthesis of secondary metabolites.