## 2 Results
### Genome Mining of the trans-PT Gene Family Identified More GFPPSs beyond the Brassicaceae Clade  
After validating the PTS-Pre by experimentally verified testing sequences, we further employed the PTS-Pre in functional annotations for _trans_-PTs from whole species. First, we used the PTS-Pre in the functional identification of _trans_-PT gene family in some specific species. Three species were randomly selected from Bacteria and Eukarya including _Streptomyces azureus_ (Actinobacteria), _Tolypothrix camylomenoides_ (Cyanobacteria), and tree cotton _Gossypium arboretum_ (Viridiplantae) for the analysis. A total of 12 sequences were applied to PTS-Pre analysis; the corresponding genes were synthesized, expressed in _E. coli_, and subjected to in vitro biochemical assays. As shown in Table 1 and Figure 3D, most of the _trans_-PTs from _T. camylomenoides_ and _G. arboretum_ were correctly predicted (prediction accuracy 87.5%, 7/8) except for _Ga_GGPPSL-4 (GI XP_017649086) and _Tc_GGPPSL-2 (GI KAB8317749). _Ga_GGPPSL-4 had no activity detected, while _Tc_GGPPSL-2 was predicted as GGPPS (C20) but finally revealed as GPPS (C10). The prediction accuracy for _S. azureus_ was low (50%, 1/2), consistent with the previous observations through SSN assignment and phylogenetic tree analysis that genes from Bacteria had the lowest prediction accuracies. To our surprise, two new GFPPSs from _T. camylonemoides_ (_Tc_GGPPSL-4) and _G. arboreum_ (_Ga_GGPPSL-3) were predicted and experimentally verified. According to the previous study, plant GFPPSs were only identified in the woody plant _Leucosceptrum cannum_ and several Brassicaceae species [10; 25; 26]. The newly identified GFPPS in _G. arboreum_ suggests that there would be more seesterrepene (C25) related genes in other plant species. Therefore, we employed the PTS-Pre tool in genome mining for searching potential GFPPSs using a previously reported dataset which contains 7870 _trans_-PTs sequences [21]. The final prediction results revealed the abundance of _trans_-PTs with different chain lengths, indicating that FPPS (C15) had the highest abundance (39% of total sequences), while GGPPS (C20) and PPPS (\(\geq\)C30) covered 24% and 32% of total sequences, respectively, and only 5% of the sequences were predicted as GFPPS (C25) (Figure 3E). We further classified these GFPPS sequences by their species, and 45 sequences were identified belonging to the plant kingdom (Dataset2). Sixteen representative sequences of the potential plant GFPPSs were synthesized and tested using in vitro biochemical assays. Finally, nine of them were identified with GFPPS activity (Figures 4A and S8, Table 2). Interestingly, all these newly identified GFPPSs were beyond the Brassicaceae clade. To explore the evolutionary distribution of GFPPSs in the plant kingdom, we generated a phylogenetic tree using all predicted plant GFPPS candidates including the nine newly identified GFPPSs. The unrooted phylogenetic tree shows that the nine newly found GFPPSs, similar to GFPPSs from the Brassicaceae clade, are also lineage-specific (Figure 4B). The nine genes could be divided into four clades, namely, Nostocaceae, Malvacae, Musaceae, and Ranunculaceae. It is noteworthy that the Nostocaceae family (which is a kind of Cyanobacteria) does not belong to the Viridiplantae kingdom but has a close evolutionary relationship with it. In the phylogenetic tree, the five GFPPS clades were located at different blanches and clearly separated. Interestingly, all the predicted GFPPSs in Peaceae clade including proteins from _Sorghum bicolor_ (XP_021311468), _Panicum hallii_ (XP_025814700), and _Setaria italica_ (RCV24163) were finally determined as GGPPS (Figure 4A,B). Taken together, these results suggest that the GFPPS genes may have been the result of convergent evolution and are more likely lineage-specific.