# Impact of Environmental Factors on Stilbene Biosynthesis
## 2 Biosynthesis of Stilbenes and Stilbenoids
### Stilbene Synthas  
The enzyme stilbene synthases (STS) catalyze the direct formation of the stilbene skeleton through a single reaction from three units of malonyl-CoA and one CoA-ester of a cinnamic acid derivative (\(p\)-coumaroyl-CoA to form _trans_-resveratrol or cinnamoyll-CoA to form _trans_-pinosylvin) [52] (Figures 2 and 3). Malonyl-CoA is generated through a carboxylation reaction between acetyl-CoA and a bicarbonate ion (HCO3\({}^{-}\)) catalyzed by acetyl-CoA carboxylase (EC 6.4.1.2) in the presence of ATP (Figure 4). Figure 2: Stilbene biosynthesis in plants. (PAL) phenylalanine ammonia- lyase; (PTAL) bifunctional L-phenylalanine/L-tyrosine ammonia-lyase; (C4H) cinnamate 4-hydroxylase; (4 CL) 4-coumarate:CoA ligase; (CNL) cinnamate: CoA ligase; (STS) stilbene synthase; (RS) resveratrol synthase; (PS) pinosylvin synthase. Figure 4: Reactions catalyzed by chalcone synthase (CHS) and stilbene synthase (STS) to produce naringenin chalcone and resveratrol, respectively. R = H phenylalanine (Phe); R = OH tyrosine (Tyr). Double arrows indicate multiple steps in the biosynthetic pathway. Figure 3: Examples of reactions catalyzed by stilbene synthase enzymes. (**A**) Conversion of \(p\)-couamoyl-CoA into \(t\)-resveratrol by resveratrol (RS) synthase (or trihydroxystilbene synthase I). (**B**) Conversion of cinnamyl-CoA into \(t\)-pinosylvin by pinosylvin synthase (PS). (**C**) Conversion of dihydro-cinnamoyl-CoA into dihydropynosylvin by dihydro-pinosylvin synthase (DPS). (**D**) Conversion of caffeoyl-CoA into \(t\)-piceatannol, probably catalyzed by PS. Based on the preferred starting substrate, STS enzymes are classified into either a _p_-coumaroyl-CoA-specific type, such as trihydroxystilbene synthase I (also known as resveratrol synthase, EC 2.3.1.95), or a cinnamoylI-CoA-specific type, such as pinosylvin synthase (EC 2.3.1.146) (Figures 2 and 3). The former type has been mainly found in angiosperms like peanut [53], grapevine [54], and Tatar rhubarb (_Rleum tataricum_ Lf) [55], while the latter type is typical in conifers and has been identified in several _Pinus_ species like Scots pine (_P. sylvestris_ L) [56], Japanese red pine (_P. densiflora_ Siebold & [PERSON].) [57], and Eastern white pine (_P. strobus_ L.) [58]. _Pinus_ species can biosynthesis two types of stilbenes, i.e., pinosylvin and dihydropinosylvin, which are biosynthetically derived from cinnamoylI-CoA and dihydrocinnamoyl-CoA, respectively (Figure 3B,C). STS from _P. strobus_ shows a clear preference for cinnamoylI-CoA and was therefore characterized as pinosylvin synthase [58]. Otherwise, STS from _P. sylvestris_ shows an unusual preference for dihydro-cinnamoyl-CoA, identifying it as a dihydropinosylvin synthase [56]. STS does not exhibit absolute substrate specificity. While showing a preference for a given substrate, the same STS enzyme can accept different cinnamic acid derivatives as starting substrates catalyzing the biosynthesis of different stilbenes. For example, the enzyme responsible for the biosynthesis of piceatannol (3,5,3\({}^{\prime}\),4\({}^{\prime}\)-tetrahydroxystilbene) has not been identified yet, however, pinosylvin synthase from _P. strobus_ proved to be active with caffeoyl-CoA in vitro (Figure 3D), suggesting that it could be responsible for piceatannol biosynthesis _in planta_[58]. STS enzymes belong to the type III polykeletic synthase superfamily (PKSs), which also includes chalcone synthase (CHS; EC 2.3.1.74) [59]. STS and CHS share a high degree of similarity both in their amino acid sequence identity (which reaches 75-90% depending on the species) and in their crystallographic structures [51; 60]. _CHS_ genes are present in the genome of all plants analyzed so far, while _STS_ have been identified in a limited number of plant species, often phylogenetically unrelated. Converging lines of evidence indicate that CHS is the archetypal enzyme from which STS evolved multiple times independently in stilbene-producing plants, through gene duplication followed by functional divergence [60; 61; 62].