A Sesquiterpene Synthase from the Endophytic Fungus _Serendipita indica_ catalyzes Formation of Viridiflorol  
Fani Ntana  
1 Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark; [EMAIL_ADDRESS]. Sesquiterpenoid (C15) biosynthesis in Basidiomycota has received increasing attention over the last decade [4; 5; 6; 7; 8]. The specific compounds are characterized by rare structures, unusually enhanced antibiotic and cytotoxic activity [9]. The first committed step in their biosynthesis is the formation of the terpenoid scaffold by the cyclization of the linear precursor farnesyl diphosphate (_E,E_-FPP, C15), a reaction catalyzed by enzymes termed sesquiterpene synthases (STSs) [10]. Initially, STSs cleave off the diphosphate moiety from _E,E_-FPP, forming a reactive carbocation, which undergoes a series of cyclizations and rearrangements. These enzymes stabilize the carbocation intermediates, until the cascade is terminated by proton abstraction, or quenching by water. Alternatively, STSs can use a secondary carbocation, formed from an _E,E_-FPP isomer, the (3R)-nerolidyl diphosphate (3_R_-NPP), and then proceed to the formation of the terpenoid skeleton (Figure 1). Interestingly, a comprehensive phylogenetic analysis of all available basidiomycete STSs showed that these enzymes are grouped in phylogenetic clades (phylogenetic clusters), not only according to sequence similarity, but also based on their cyclization mechanism [11]. In detail, all the enzymes of each phylogenetic clade (Figure 2) catalyze the same first carbon-carbon (C-C) bond forming reaction (e.g., Clade II enzymes perform 1,10 cyclization of (3R)-NPP) (Figure 1) [3]. This means that a simple phylogenetic analysis can offer a predictive framework for targeted discovery of sesquiterpenoids with a specific skeleton [11]. Figure 1: Proposed biosynthetic pathways of several sesquiterpenes found in basidiomycete fungi. Sesquiterpenoid scaffolds (and the respective STSs) can be distinguished by the first cyclization step of _E,E_-FPP or (3_R_)-NPP. Here, four distinct structural groups are presented in different colors, each one containing terpenids formed by different types of STSs. Terpenoids derived from 1,10 closure of _E,E_-FPP are shown in pink (STS Clade I), 1,11 closure of _E,E_-FPP are shown in red (STS Clade III), 1,6 or 1,7 closure of (3_R_)-NPP are shown in yellow (STS Clade IV) and 1,10 closure of (3_R_)-NPP are shown in blue (STS Clade II) (adapted from [3]). _S. indica_ (formerly known as _Piriformospora indica_) is an endophytic fungus, belonging to the phylum Basidiomycota and extensively used in plant-fungal interaction studies [16; 17; 18]. The endophyte can successfully colonize several plant species as a root mutualist [19], conferring several benefits to the host plant, including increased plant biomass [20; 21; 22; 23], and improved plant resistance against biotic [24; 25; 26; 27] and abiotic challenges [20; 28; 29]. _S. indica_ has a characteristic biphasic colonization style, during which a transition from an initial biotrophic growth to a later plant cell-death associated phase is observed [26]. To colonize plant roots successfully, _S. indica_ suppresses host immune responses by secreting effectors and interfering with the plant hormone biosynthesis and signaling [26; 30; 31]. In addition, the endophyte is able to produce phytohormone analogues that contribute further to the establishment of the mutualistic interaction with the host [32; 33]. Colonization by _S. indica_ was also shown to stimulate host specialized metabolism in several plants [34; 35; 36], but until now there has been no evidence that this endophytic fungus can itself produce any type of specialized metabolite. In fact, sequencing of the _S. indica_ genome showed that the endophyte lacks the large repertoire of terpenoid biosynthetic genes that characterize other basidiomycetes [9]. Here, we describe the discovery and the functional characterization of a terpene synthase gene (_SiTPS_), found in the genome of _S. indica_.