# Biological Properties of Sandalwood Oil and Microbial Synthesis of Its Major Sesquietrepenoids
## 5 Metabolic Engineering Strategies to Increase the Yield of Santalene and Santalol in S. cerevisiae
### Engineering of the Key Enzymes in the MVA Pathway
\begin{table}
\begin{tabular}{c l l l l l l} \hline \hline
**Host** & **Metabolic Engineering Strategy** & **Santalene** & **Redox** & **Fermentation** & **Yield** & **Ref.** \\
**Organism** & **Metabolic Engineering Strategy** & **Synthase** & **System** & **Scale** & & **[67]** \\ \hline \multirow{4}{*}{_S. cerevisiae_} & Altering ERG9 expression by & & & & & 13 g/L santalol & \\  & replacing promoter with PHXT1; & & SaSS and & CYP736A167- & Fed-batch, 5 L & 1.2 g/L & \\  & overexpression of santalol biosynthesis genes under GAL & & CISS & SaCPR2 & bioreactor & Z-\(\alpha\)-santalol & \\  & promotors. & & & & & \\ \hline \multirow{4}{*}{_S. cerevisiae_} & Integration of optimized & & & & 164.7 mg/L & \\  & P450-CPR redox system for & & & & & \\  & santalols production; & & & 464 ATR1 opt & 100 mL shake & santalene & \\  & downregulating ERG9 gene. & & & & & \\ \hline \multirow{4}{*}{_S. cerevisiae_} & Optimization of precursor and & & & & & \\  & cofactor supply; optimization of & & & & & \\  & the FPP branch point; modulation & & & & & \\  & of the MVA pathway; & & & & & \\  & modification of the ammonium & & & & & 0.036 Cmmol (g & \\  & assimilation pathway; enhancing & & & & & of \(\alpha\)-santalene & \\  & the activity of a transcriptional & & & & & \\  & activator; continuous fermentation & & & & & \\  & process optimization. & & & & & \\ \hline \multirow{4}{*}{_Y. lipolytica_} & Overexpression of MVA pathway & & & & & & \\  & genes; optimization of glucose & & & & & \\  & concentration; fermentation & & & & & \\  & optimization through genetic and & & & & & \\  & feeding strategies. & & & & & \\ \hline \multirow{4}{*}{_E. coli_} & Amplified flux toward FPP & & & 100 mL shake & 1272 mg/L & \\  & precursor; engineered santalene & & & & & \\  & synthase through mutagenesis & & & & Fed-batch 1.3 L & 2916 mg/L & \\  & and fusion tag. & & & & bioreactor & \(\alpha\)-santalene & \\ \hline \multirow{4}{*}{_E. coli_} & Manipulated ribosome binding & & & & & \\  & sites (RBSs) to optimize synthetic & & & & & \\  & operon; deleted tnA gene to & & & & & \\  & increase \(\alpha\)-santalene production & & & & & \\ \hline \multirow{4}{*}{_Komagataella_} & Promoter optimization; gene & & & & & 829.8 mg/L & \\  & overexpression; multi-copy & & & & & \\  & integration for biosynthesis & SaSS & - & & & \\  & rewiring; medium optimization; & & & & & \\  & bioprocess engineering. & & & & & \\  & & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Progress in santalene and santalol biosynthesis and metabolic engineering strategies used. including ergosterol, ubiquinone, etc., where the overexpression effectiveness of the ERG20 gene to maximize the FPP yield depends on the yeast strain used and culturing conditions [96; 97]. It has been shown that integrating and over-expressing the genes glutamate dehydrogenase GDH2 and ERG20 significantly improved sesquiterpene synthesis, subsequently maximizing the \(\alpha\)-santalene yield. Also, the \(\alpha\)-santalene yield was improved upon further up-regulation of these two genes [88]. Furthermore, integrating and over-expressing the mutant transcription factor gene and an additional supply of truncated HMG1 into the yeast genome improves the effectiveness of the MVA pathway [52].