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  • Over decades a deeper understanding of terpene

    2022-05-23

    Over decades, a deeper understanding of terpene synthases has also permitted enzyme engineering for unnatural substrate acceptance. Novel terpenoids have been successfully synthesized from substrate analogues as by-products from mechanistic investigation (Faraldos et al., 2012; Miller, Yu, & Allemann, 2007; Miller, Yu, Knight, & Allemann, 2009) or for biological applications such as (S)-14,15-dimethylgermacrene D by (S)-germacrene D synthase, a powerful aphid attractant (Cascón et al., 2012; Touchet et al., 2015). A pheromone used by destructive pine beetles, (+)-verbenone, was also synthesized in one pot from geranyl pyrophosphate (Yoosuf-Aly, Faraldos, Miller, & Allemann, 2012). Here we describe how to express and purify one of the numerous sesquiterpene synthases and then employ it to catalyze the formation of a new sesquiterpenoid. We chose germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor to illustrate the procedure. GdolS is a 315 amino Sertraline HCl australia bacterial sesquiterpene cyclase that catalyzes the conversion of FDP to germacradien-4-ol (6) as the sole product (Grundy et al., 2016). Recently, we have shown that GdolS also accepts other substrates such as 10,11-epoxy-FDP (7), which it turns over to the 11-membered cyclic ether 8 (Fig. 2) (Huynh, Grundy, Jenkins, Miller, & Allemann, accepted).
    Sesquiterpenes Production and Purification
    Enzymatic Synthesis of Cyclic Ether After the successful expression of the sesquiterpenes synthase, the recombinant enzyme can be used to catalyze the conversion of a selected substrate to a sesquiterpenoid. Conversion of 10,11-epoxy-FDP by GdolS will be illustrated in this chapter. When using an unnatural substrate, it is advised to always test the activity of the enzyme against its natural substrate. This procedure is described in Section 3.1.
    Conclusion Over the last decade, numerous novel sesquiterpenoids have been synthesized using chemoenzymatic processes with modified substrates. FDP substrates modified with hydroxy, fluoro, or methyl groups not only result in new compounds but can also provide access to novel activities, as seen with (14,15-dimethyl)-GDS (Touchet et al., 2015). Often chemoenzymatic approaches to terpenoids allow a shortening of the synthetic pathway as was the case for the production of dihydroartemisinic aldehyde, a key intermediate for the generation of artemisinin (Demiray et al., 2017). The heterologous expression of germacradien-4-ol synthase in E. coli allowed us to screen a variety of modified substrates. One of these generated an unusual cyclic ether, a linkage currently not known to be formed as a direct product of terpene cyclases in nature; it represents an entirely novel expansion of the terpenome. The use of unnatural substrates with sesquiterpene synthases is an elegant route to create new structures in a cost-effective and ecologically friendly manner. In our hands, this method has already been applied to aristolochene synthase, germacrene A synthase, and δ-cadinene synthase to yield ethereal products. This methodology may prove useful for discovering novel biologically active compounds and for easy access to chiral building blocks.
    Introduction The genes encoding for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), farnesyl diphosphate synthase (FPPS), and alpha-farnesene synthase (AFS) are involved in the formation of alpha-farnesene (Lurie and Watkins, 2012, Rupasinghe et al., 1998). When lovastatin, a competitive inhibitor of HMG-CoA reductase, was used to treat fruits, the formation of alpha-farnesene and the incidence of apple superficial scald were almost completely blocked (Ju and Curry, 2000). This indicated that it is possible to control apple superficial scald by controlling the genes involved in the formation of alpha-farnesene. As a first step toward a better understanding of the role of HMGR, FPPS and AFS in apples, we have reported some research results of the AFS gene and the FPPS gene (Yuan et al., 2007, Yuan et al., 2009, Yuan et al., 2010, Yuan et al., 2011). Now, we report our new results on the FPPS gene in apples.