Biosynthesis of Sodorifen, Chlororaphen and Related Compounds

索多利芬和氯代菲是细菌来源的非典型甲基化萜类。它们的生物合成将通过克隆和异源表达分别在粘性沙雷氏菌和绿假单胞菌生物合成基因簇中编码的基因来进行。在花斑变种(Variovorax Boronicumulans)和格氏假单胞菌(Pseudomonas Grimontii)中发现了两个相关的生物合成基因簇，它们的基因与Sodorifen和氯代环烯生物合成基因簇中观察到的基因同源，但这些基因簇的产物尚不清楚。此外，对于这些簇，酶表达构建体将可用于酶促产生未知产物，用于分离和结构阐明。用同位素标记的前体进行孵化实验，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。甲基转移酶和非典型性萜烯合成酶参与大豆异构体和氯代环烯生物合成的结构尚不清楚。这些酶将与其天然底物的非反应性类似物一起结晶，并将被合成。基于所获得的晶体结构，将进行定点突变实验，以更深入地了解酶的作用机制。带有额外甲基化的底物类似物将被合成，并与萜烯合成酶进行酶促转化，以合成Sodorifen和氯化环烯。得到的产物将被提纯，其结构将被阐明。综上所述，该项目旨在从机理上深入了解与Sodorifen、氯代菲和相关分子的生物合成有关的不寻常的酶。

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.