## 2 Results and Discussion
### Identification of the Sesquiterpene Products Catalyzed by TaTS9  
The purified _Ta_TS9 (100 \(\upmu\)g/mL) and FPP (100 \(\upmu\)m) were incubated at 50 rpm and 28 \({}^{\circ}\)C overnight, and the sesquiterpene products were extracted by hexane and identified by the GC-MS. As shown in Figure 7b, three peaks with the retention time at 18.462 min, 18.569 min, and 19.394 min were detected, corresponding to \(\upbeta\)-cubenene, \(\upbeta\)-cubenene, and \(\upgamma\)-amorphene, respectively. The chromatogram revealed the presence of cyclic sesquiterpenes with typical mass fragmentation patterns at 105, 119, 161, and 204 m/z, without and with the hydroxyl group (Supplementary Figure S7). The major peaks were predicted to be the large amounts of \(\upbeta\)-cubenene isomers (>90%) and tiny \(\upgamma\)-amorphene, respectively. As a negative control, no terpenoids were detected in the reaction system containing only FPP. Therefore, _Ta_TS9 was then classified as a sesquiterpene synthase that catalyzes the cyclization of FPP to produce \(\upbeta\)-cubenene.  
Cubenene synthase is common in plants like _Pyrus_ spp. that catalyze the biosynthesis of volatile cubenene [29]. Its existence in fungi was reported by [PERSON] et al. [29] for the first time, in which a (+)-cubenene synthase was identified in _A. chrysogenum_. Due to the very low sequence similarity of fungal sesquiterpene synthases, such as the <30% similarity between _Ta_TS9 and _AcTPS3_ and 25.82-47.71% similarity of seven _Ta_TSs to known Swiss-Prot homologues (Figure 1), it is inaccurate to predict the functions of new sesquiterpene synthases [20; 30]. Moreover, the low yield of sesquiterpenes obtained through direct extraction from the native host, especially the volatile sesquiterpenes, hinders the pace of biochemical characterization. Synthesis of new products through biological instead of chemical approaches is more environmentally friendly and sustainable. Heterologous expression of terpene synthases in engineering strains is more feasible to increase the yields of terpenoids. In the case of _Ac_TPS3, cubenene achieved the highest titer of 597.3 mg/L in the optimized _S. cerevisiae_ so far [28]. However, the application potential of cubenene has  
Figure 7: Purification of the recombinant terpene synthase _Ta_TS9 (**a**) and product analysis by GC-MS using FPP as the precursor (**b**). and the structure and mass spectra of \(\upbeta\)-cubenene (**c**) and \(\upgamma\)-amorphene (**d**). Lanes: M, marker; 1, the culture supernatant; 2–9, the eluates of 0, 20, 40, 100, 150, 200, 250 and 300 mM imidazole, respectively.  
not been assayed yet. In contrast, we identified _TaTS9_ as the key gene in the biosynthesis of anti-fungal sesquiterpenoids through genome mining, antagonism assay, transcript quantification, and VOC metabolome analysis, and we determined its products to be mainly \(\beta\)-cubenene. However, due to the low yield in the present biosynthesis system, the biological activities of \(\beta\)-cubenene against plant pathogens were unassessed. Besides the MVA pathway of _S. cerevisiae_, an isopentenol utilization IU pathway-dependent (IUPD) strain of _S. cerevisiae_ has been developed, which surpassed the MVA pathway in the growth-coupled production of complex terpenoids [31]. Thus, our future studies would focus on the improvement and optimization of the \(\beta\)-cubenene biosynthesis system for application in the agriculture industry.