## 3 Discussion  
In this study, we provided the first cloning and functional characterisation of _Pm_STPS1 encoding a putative \(\beta\)-farnesene synthase, and _Pm_STPS2 encoding a putative nerolidol synthase in _P. minus_. A sequence comparison between the _Pm_STPS1 and _Pm_STPS2 indicated that the two enzymes had different protein and nucleotide sequences. However, both of the enzymes were structurally similar to other plant sesquiterpene synthases and contained all of the conserved motifs, including DDxxD, RXR, and NSE/EDTA, which were important for terpene synthase functionality [5; 45; 46]. Based on the phylogenetic analysis, _Pm_STPS1 and _Pm_STPS2 clustered on the same group with four distinct sesquiterpene synthases. In addition, the unexpected band in the upper layer (Figure S4) might have been caused by protease aggregation, according [47]. As shown in several recent studies, the protein sizes of a few plants sesquiterpene synthases showed a nearly similar molecular weight, with _Pm_STPS2 within the range of 60-70 kDa [48; 49; 24]. In general, terpene synthase could be classified into monoterpene, sesquiterpene, and diterpene synthase, with 550-860 amino acids encoding a 50-100 kDa protein [4; 50]. As a result of the absence of the signal peptide sequence with 50-70 amino acids, the size of sesquiterpene synthases were typically smaller than those of the monoterpenes and diterpenes [51]. [PERSON] et al. [37] also reported that the protein size of the _P. minus_\(\beta\)-sesquiphelandrene synthase was 65.1 kDa. Additionally, studies on \(\beta\)-caryophyllene synthase, that were encoded by OkBCS (GenBank accession no. KP226502) from _Ocinum killimandscharicum_ Gurke, showed a molecular weight of 63.6 kDa [24]. Until recently, no short sesquiterpene synthase sequence was characterised as a _Pm_STPS1. However, the short-chain length of this enzyme could be associated with several prenyltransferase (PT) enzymes. Based on previous findings, two prenyltransferase, Santalum farnesyl diphosphate synthase (SaFDS) and Hedychium farnesyl pyrophosphate synthase (HcFPPs), comprising 1029 and 1068 bp nucleotide sequences and encoding polypeptides of 343 and 356 amino acids, respectively, were reported [3; 52]. Figure 3: GC-MS chromatogram of products formed by crude _Pm_STPS1 and _Pm_STPS2 protein at different retention times (RT). (**a**) The chromatogram of control used consisted of M15 _E. coli_ host harboring empty pQE-2 taqzyme plasmid; (**b**) _Pm_STPS1 (**c**); and _Pm_STPS2. Many terpene synthases (TPSs) had the ability to synthesise one or multiple products from a single substrate, regardless of whether it was farnesyl pyrophosphate (FPP) or geranyl pyrophosphate (GPP) [53; 54]. In addition, the sesquiterpene synthases from different plant species produced more than one product [55; 56; 25]. Interestingly, although the sizes of _Pm_STPS1 and _Pm_STPS2 were different, the two enzymes produced similar sesquiterpene products (\(\beta\)-farnesene, \(\alpha\)-farnesene, and farnesol, Figure 3), albeit at different percentages. Both _Pm_STPS1 and _Pm_STSP2 could catalyse the formation of \(\beta\)-farnesene, \(\alpha\)-farnesene, meridold, and farnesol, whose functions were different from those of the previous STPS enzymes characterised in _P. minus_[36; 37; 38; 39]. In addition, the enzymes demonstrated an inherent capacity for TPS enzymes to evolve different products and substrate specificities [57]. Moreover, the main factor of sesquiterpene diversity was the large number of different sesquiterpene synthases expressed in plants and the ability of some sesquiterpene synthases to form multiple products from a single FPP substrate [58]. Based on previous chemical profiling studies of _P. minus_ essential oils from hydro-distillation extraction, low levels of meridold and farnesol were detected at 0.24% and 0.14%, respectively [34]. A similar finding was reported, which indicaed that the percentage of \(\beta\)-farnesene and \(\alpha\)-farnesene compounds were also found at 0.92% and 0.82%, respectively [31; 32; 34].