Biosynthesis of Sodorifen, Chlororaphen and Related Compounds

索多芬、氯莱芬及相关化合物的生物合成

• 批准号: 534926425
• 负责人: Professor Dr. Jeroen Dickschat
• 金额: --
• 依托单位: Kekulé-Institut für Organische Chemie und Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/534926425?language=en
• 关键词: Biosynthesis; Sodorifen; Chlororaphen; Related; Compounds

Sodorifen and chlororaphen are non-canonical methylated terpenes of bacterial origin. Their biosynthesis will be studied by cloning and heterologous expression of the genes encoded in biosynthetic gene clusters in Serratia plymuthica and Pseudomonas chlororaphis, respectively. Two related biosynthetic gene clusters containing homologous genes to those observed in the biosynthetic gene clusters of sodorifen and chlororaphen are found in Variovorax boronicumulans and Pseudomonas grimontii, but the products of these gene clusters are unknown. Also for these clusters enzyme expression constructs will be made available to enzymatically generate the unknown products for isolation and structure elucidation. Incubation experiments with isotopically labelled precursors will be performed to elucidate the complex cyclisation mechanisms and to determine the absolute configurations of the methylated terpenoids. This work will be supported by DFT calculations. The structures of the methyltransferases and the non-canonical terpene synthases involved in sodorifen and chlororaphen biosynthesis are unknown. These enzymes will be crystallised together with non-reactive analogs of their native substrates that will be synthesised. Based on the obtained crystal structures site-directed mutagenesis experiments will be performed to gain deeper insights into the enzyme mechanisms. Substrate analogs with additional methylations will be synthesised and enzymatically converted with the terpene synthases for sodorifen and chlororaphen biosynthesis. The obtained products will be purified and their structures will be elucidated. Taken together, this project aims at a deep mechanistic understanding of the unusual enzymes involved in the biosynthesis of sodorifen, chlororaphen and related molecules.

Soddophen和chlororaphen是细菌来源的非典型甲基化萜烯。它们的生物合成将通过克隆和异源表达的基因编码的生物合成基因簇中的沙雷氏菌和绿针假单胞菌，分别进行研究。在Variovorax boronicumulans和Pseudomonas grimontii中发现了两个相关的生物合成基因簇，其中含有与在sodorifen和chlororaphen的生物合成基因簇中观察到的基因同源的基因，但这些基因簇的产物尚不清楚。同样对于这些簇，酶表达构建体将可用于酶促产生未知产物以用于分离和结构解析。将进行同位素标记的前体的孵育实验，以阐明复杂的环化机制，并确定甲基化萜类化合物的绝对构型。这项工作将得到DFT计算的支持。甲基转移酶和参与钠草醚和氯草芬生物合成的非典型萜烯脱氢酶的结构是未知的。这些酶将与将要合成的其天然底物的非反应性类似物一起结晶。基于获得的晶体结构，将进行定点突变实验，以更深入地了解酶的机制。将合成具有额外甲基化的底物类似物，并用萜烯糖苷酶进行酶促转化，用于钠草醚和氯草芬生物合成。所得产物将被纯化，并对其结构进行解析。总之，该项目旨在深入了解参与sodorifen，chlororaphen和相关分子生物合成的不寻常酶的机理。