## 4 Common Strategies for Optimizing Sesquietpenol Biosynthesis
### Enzyme Engineering  
#### 4.1.1 Identification and Functional Verification of Key Enzymes  
The biosynthesis and utilization of farnesyl pyrophosphate (FPP) are pivotal factors that directly influence the final titers of sesquiterpenol production. The rapid advancement of DNA sequencing technologies has made genetic sequence information available in public databases. This enables the identification of potential gene sequences for heterologous expression in microbial chassis, which can achieve high-yield production (Figure 4a, Table 2). The crucial genes identified thus far that are involved in sesquiterpenol biosynthesis are \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Classification** & **Sesquiterpenol** & **Host** & **STRATEGIES** & **Scales** & **Titers (mg/L)** & **Reference** \\ \hline \hline \multirow{8}{*}{patchoul} & \multirow{8}{*}{_Saccharomyces cerevisiae_} & fusion of farnesyl & & & & \\  & & & pyrophosphate (FPP) synthase & & & \\  & & & and patchoul synthase to & & & \\  & & & increase the utilization of FPP & & & \\  & & & precursors; enhanced the & & & \\  & & & expression of rate-limiting & & & \\  & & & genes in the meyanolate & & & \\  & & _cerebisae_ & pathway; squalene synthase & & & \\  & & & was weakened by the & & & \\  & & & glucose-inducible promoter of & & & \\  & & & HXT1 to reduce the metabolic & & & \\  & & & flux from FPP to ergosterol; & & & \\  & & & inhibited the biosynthesis of & & & \\  & & & farnesol, thereby reducing the & & & \\  & & & consumption of FPP & & & \\ \hline \hline \multirow{8}{*}{patchoul} & \multirow{8}{*}{_Saccharomyces cerevisiae_} & fusion of farnesyl & & & & \\  & & pyrophosphate (FPP) synthase & & & & \\  & & and patchoul synthase; & & & & \\  & & introducing the patchoul & & & & \\  & & biosynthesis pathway into the & & & & \\  & & mitochondria & & & & \\  & & mitochondria & & & & \\  & & & & & \\  & & & & & & \\  & & & & & & \\ \hline \multirow{8}{*}{Polycyclic} & \multirow{8}{*}{_Pichia pastoris_} & fusion of farnesyl & & & & \\  & & pyrophosphate (FPP) synthase & & & & \\  & & and patchoul synthase; & & & & \\  & & increased precursor supply; & & & & \\  & & added auxiliary carbon source & & & & \\ \hline \multirow{8}{*}{patchoul} & \multirow{8}{*}{_Yarrowia lipolytica_} & downregulated squalene & & & & \\  & & synthesis based on & & & & \\  & & Cu\({}^{2+}\)-repressible promoter; & & & & \\  & expanded the meyanolate & & & & & \\  & precursor pool; fusion of & & & & & \\  & & farnesyl pyrophosphate (FPP) & & & & \\  & & synthase and patchoul & & & & \\  & & synthase & & & & \\ \hline \multirow{2}{*}{santalo} & \multirow{2}{*}{_Saccharomyces cerevisiae_} & downregulated ERG\({}^{\prime}\) gene; & & & & \\  & & truncated the N-terminal 46 & & & & \\  & & amino acid of ATRI & & & & \\ \hline \multirow{8}{*}{santalo} & \multirow{8}{*}{_Saccharomyces cerevisiae_} & replaced the natural ERG\({}^{\prime}\) & & & & \\  & & promoter with the tunable & & & & \\  & & HXT1 promoter; & & & & \\  & & overexpressed smalla & & & & \\  & & synthesis pathway; & & & \\  & & overexpressed GAL\({}^{\prime}\) (a & & & \\  & & transcriptional activator of & & & & \\  & & _GAL_ promotions) and PCM2 (a & & & & \\  & & yeast phosphoglucumatase) & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: _Cont._listed in Table 3. To date, nerolidol synthases (NES) have been identified from five distinct plant species [65; 83; 84; 85; 86], with functional validation achieved in _Saccharomyces cerevisiae_ for enzymes derived from _Actinidia chinensis_ and _Celastrus angulatus_. [PERSON] et al. pioneered the identification of the nerolidol synthase AcNES1 from _A. chinensis_ flowers [83], while [PERSON] et al. characterized two NES isoforms (CaNES1 and CaNES2) from _C. angulatus_. Subsequent metabolic engineering strategies integrating these genes into _S. cerevisiae_ resulted in engineered strains with enhanced nerolidol yield [65]. Notably, significant interspecies variability in enzymatic properties has been observed. The AcNES1 gene from _A.