## 4 Discussion  
_S. pararoseus_ is an unconventional fungus that produces a high yield of carotenoids, which are fat-soluble isoprenoids. Our previous study showed that it accumulates three carotenoids: \(\beta\)-carotene, torulene and torularhodin [16, 20, 30]. Humans cannot synthesize carotenoids by themselves, and the demand for natural anti-radiation and anti-aging products is increasing. Therefore, it is necessary to study the carotenoid synthesis pathway. The product FPP, catalyzed by FPPSs, is used as a constituent of primary metabolism compounds such as carotenoids and steroids [24]. Research on FPPSs has been pursued for many years, but the synthesis and mechanism of FPPS in _S. pararoseus_ remain unclear. We cloned a novel cDNA (_SpFPPS_) from _S. pararoseus_. TLC analysis showed that SpFPPS had activity in vitro. SpFPPS synthesized the sole product FPP regardless of the allylic substrate (DMAPP or GPP) that was utilized. The functions of FPP synthases had previously been used to study _Anthononus grandis_, and the major product of AgFPPS was found to be FPP using DMAPP with IPP as substrates [31]. In fungus, FPP accounts for 75% of FPPS final products in _Saccharomyces cerevisiae_[32]. In _Xanthophyllomyces dendorhous_, extracts of XdFPPS formed C10 and C15 allylic pyrophosphates [33]. Consistent with SpFPPS, they can use DMAPP or GPP as substrates, but in _Mycobacterium tuberculosis_, only geranyl diphosphate served as the MtFPPS acceptor [34]; the difference is that the enzyme activity product of SpFPPS is only FPP. FPP provided a precursor for downstream synthesis of GGPP and other metabolic pathways, and most SCIPPSs catalyzed one specific product. However, some SCIPPSs have been reported to have two or even three enzyme activities (Table 1), and their products are also different. Perhaps the functional diversity of enzymes is to be attributed to their structural differences or is related to their long-term evolution. Furthermore, the reason why multifunctional enzymes prefer the above species to other organisms is still unclear and needs further exploration. Comparing the CLD of SpFPPS with that of other species, it was shown that the structural characteristics of SpFPPS were consistent with the FPPSs of birds and humans (Figure 1A). According to the characteristics of CLD, SpFPPS was classified as a type I FPPS. The phylogenetic tree of SpFPPS with the FPPSs of other organisms obtained from the NCBI was constructed by the neighbor-joining method (Figure 1B). The tree had two branches, involving two independent non-homologous isoprenoid biosynthesis metabolic pathways: \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Enzymes** & **Organism** & **Products** & **Reference** \\ \hline FPPS & _Zea mays_ L. & FPP, GGPP & [35] \\ FPPS & _Myzus persiae_ & GPP, FPP & [36] \\ GPPS & _Phalanepis mellina_ & GPP, FPP & [37] \\ GPPS & _Pica abs_ (Norway & GPP, FPP, GGPP & [38] \\ GPPS & spruce) & _Pica abs_ & GPP, GGPP & [39] \\ GPPS & _Lithospermium_ & GPP, FPP & [40] \\ FPPS & _Plasmodium falciparum_ & FPP, GGPP & [41] \\ FPPS & _Toxoplasma gondii_ & FPP, GGPP & [42] \\ GGPPPS & _Haematococcus_ & GPP, FPP, GGPP & [29] \\ SpFPPS-Y90A & _S. pararoseus_ & FPP, GGPP & This study \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Functional diversity of isoprenyl diphosphate synthases. the mevalonate pathway (MVA) and the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate pathway (DOXP/MEP) [43, 44, 45]. A branch of the phylogenetic tree was type I FPPSs, where the _Rhodotorula toruloides_ FPPS was most similar to SpFPPS. SpFPPS had significant homology with _R. toruloides, Phaffia rhodozyma_ and _Sanghuangporus baunii_, all of which belonged to the type I FPPSs. They had similar characteristics and mechanisms of chain length determination. Another branch contained type II and type III FPPSs. The FPPSs in _E. coli_ and _Bacillus stearothermophilus_ are type II FPPSs. [PERSON] et al. identified a type III \(\omega\),E,E-farnesyl pyrophosphate synthase from _M.