## 2 Results and Discussion
### Production and Purification of the Recombinant TaTS9  
The deduced _TaTS9_ contains 375 amino acid residues with a _p_l value of 6.12 and a molecular weight of 42.04 kDa, and it shares 34.06% similarity with the functionally characterized and structurally resolved longiboreenol synthase of _Fusarium graminearum_ (IIS104.1). The recombinant plasmid pET28-_TaTS9_ was transformed into _E. coli_ BL21 (DE3) and cultured overnight, followed by 20 h of induction with 0.5 mM IPTG. The cells were harvested, resuspended, and disrupted, and the crude enzyme was subjected to Ni-NTA affinity purification and ultraconcentration. As shown in Figure 7a, the purified recombinant _TaTS9_ showed a single band of 44 kDa on the SDS-PAGE, which was similar to the theoretical weight of _TaTS9_ plus His\({}_{6}\)-TEV. Although the recombinant _TaTS9_ was  
Figure 6: Venn diagram of the VOCs of the chassis strain _\(\Delta\)Talig4:neo_ and the two _TaTS_ gene knockout mutant strains _\(\Delta\)Talig4:neo_\(\Delta\)_TaTS8:hpl_ and _\(\Delta\)Talig4:neo_\(\Delta\)_TaTS9:hpl_.  
\begin{table}
\begin{tabular}{l l l l l l l l} \hline \hline \multirow{2}{*}{**No.**} & \multirow{2}{*}{**Compound**} & \multirow{2}{*}{**Retention**} & \multirow{2}{*}{**Formula**} & \multirow{2}{*}{**Molecular**} & \multicolumn{2}{c}{**Absolute Amount**} & \\  & & & & & & & \(\Delta\)_Talig4:neo_ & \(\Delta\)_Talig4:neo_ & \(\Delta\)_Talig4:neo_ \\ \hline \multirow{3}{*}{33} & (S,E)-8,12,15,15-Tetramethyl- & & & & & \(\Delta\)_Talig4:neo_ & \(\Delta\)_TaTS8:hpl_ & \(\Delta\)_TaTS9:hpl_ \\  & & pentadeca-7,11-diene & & & & & & \\ \hline
34 & 1-Octadecanol & 26.879 & C18H38O & 270.50 & \(2.76\times 10^{5}\) & \({}^{\star}\) & \({}^{\star}\) \\ \hline
35 & Panaxene & 26.935 & C15H24 & 204.35 & \(1.24\times 10^{5}\) & \(1.24\times 10^{5}\) & \({}^{\star}\) \\ \hline
36 & Methyl stearate & 27.293 & C19H38O2 & 298.50 & \(1.69\times 10^{5}\) & \({}^{\star}\) & \({}^{\star}\) \\ \hline \multirow{3}{*}{37} & Bicyclolg[9.3.1]pentadeca-3,7- & & & & & & \\  & dien-12-ol, & & & & & & \\  & 4,8,12,15,15-pentamethyl-, & & & & & & \\  & [1R-(1R*3E,7E,11R*,12R*)]- & & & & & & & \\ \hline
38 & Hexanedio acid, & & & & & & \\  & bis(2-ethylhexyl) ester & 30.678 & C22H42O4 & 370.60 & \(6.34\times 10^{5}\) & \(6.71\times 10^{4}\) & \(6.31\times 10^{4}\) \\ \hline
39 & Cholesteryl formate & 36.302 & C28H46O2 & 414.70 & \(5.56\times 10^{5}\) & \({}^{\star}\) & \({}^{\star}\) \\ \hline
40 & Cholestera-2,4-diene & 37.471 & C27H44 & 368.60 & \(5.17\times 10^{5}\) & \({}^{\star}\) & \({}^{\star}\) \\ \hline
41 & Cholester-5-en-3-ol & & & & & & \\  & (3.beta.)-, nonanoate & 37.909 & C36H62O2 & 526.90 & \(1.74\times 10^{6}\) & \({}^{\star}\) & \({}^{\star}\) \\ \hline Total & & & & & & \(1.88\times 10^{8}\) & \(4.34\times 10^{7}\) & \(5.29\times 10^{7}\) \\ \hline \hline \end{tabular}
\end{table}
Table 3: _Cont._successfully produced in the _E. coli_, the yield was low, which might be due to the prokaryotic incompatibility. However, the heterologous expression system of _E. coli_ is always preferred for the functional characterization and product identification of TSs due to the absence of the mevalonate (MVA) pathway and the simpleness of the reaction system consisting of the precursor FPP and recombinant TS. For instance, the terpenoid cyclase _Ta_TC6 from _T. atroviride_ FKI-3849 produced in _E. coli_ BL21 (DE3) could convert FPP into trichobrasilenol with some sesquiterpenes by-products [20].