Biosynthesis of Sodorifen, Chlororaphen and Related Compounds

索多利芬和氯芬是细菌来源的非规范甲基化萜。它们的生物合成将分别通过克隆和异源表达在增粘沙雷菌和绿假单胞菌的生物合成基因簇中编码的基因进行研究。在boronicumulans和Pseudomonas grimontii中发现了两个与sodorifen和chlororphenen的生物合成基因簇含有同源基因的相关基因簇，但这些基因簇的产物尚不清楚。此外，对于这些簇，酶表达结构将可用于酶促生成未知产物进行分离和结构解析。将进行同位素标记前体的孵育实验，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。甲基转移酶和非规范萜烯合成酶参与索多利芬和氯芬的生物合成的结构是未知的。这些酶将与将被合成的天然底物的非反应性类似物一起结晶。基于所获得的晶体结构，将进行位点诱变实验，以更深入地了解酶的机制。具有额外甲基化的底物类似物将被合成并与萜类合成酶酶促转化，用于索多利芬和氯酚的生物合成。所得到的产物将被纯化，其结构将被阐明。综上所述，该项目旨在深入了解参与索多利芬、氯芬和相关分子生物合成的不寻常酶的机制。

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.