# Biological Properties of Sandalwood Oil and Microbial Synthesis of Its Major Sesquietrepenoids
## 3 Biosynthesis of Santalene, Santalol, and Other Terpenoids Found in Sandalwood Oil  
Despite the vast structural diversity of terpenoids, they all originated from two 5-C molecules, namely, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are naturally produced via the mexulonic acid (MVA) pathway and non-mesulonic acid pathway also known as 2-C-methyl-D-erythtrol-4-phosphate (MEP) pathway (Figure 2) [48; 49; 50]. The MVA pathway, native to plant cytosol and eukaryotes, has been successfully integrated into yeast [51; 52] and bacteria [53]. Six enzymic catalytic steps (Table 2) direct carbon flux from acetyl-CoA toward IPP formation. Several studies have shown that the enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), mexulonate kinase (MK), phosphomevalonate kinase (PMK), and isopentenyl diphosphate isomerase (IDI) are considered to be rate-limiting steps [54; 55]. Conversely, a non-mesulonic acid or MEP pathway is native to plant plastids and most eubacteria. Seven enzymic catalytic steps (Table 2) are involved in the formation of IPP and DMAPP from pyruvate and glyceraldehyde-3-phosphate (G3P), whereby it has been shown that four enzymes, 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 2-C-methyl-D-erythtrol 4-phosphate cytidylyltransferase (ispD), 2-C-methyl-D-erythtrol-2,4 cyclodiphosphate (ispF), and isopentenyl diphosphate isomerase (IDI), are involved in what is known as the rate-limiting step in _E. coli_[56; 57]. Also, the isopentenol utilization pathway (IUP) has recently become an alternative metabolic pathway to increase the supply of DMAPP or IPP in recombinant strains. This pathway comprises promiscuous kinase and isopentenyl phosphate kinase, which consecutively phosphorylate prenol into DMAPP via the intermediate dimethylallyl phosphate (DMAP). Promiscuous kinases can be combined with isopenten \begin{table}
\begin{tabular}{l c c c} \hline \hline
**Pathway** & **Starting Carbon Source** & **Enzyme Catalyzed Steps** & **Precursor Formed** \\ \hline MVA pathway & Acetyl-CoA & 6 & IPP \\ \hline MEP pathway & Pyruvate and GSP & 7 & IPP and DMAPP \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Comparison of biosynthesis pathways for generating terpenoid precursors. provide a simple and effective enzymatic approach for manufacturing DMAPP in vitro, employing prenol as a suitable substrate and ATP as a cofactor [58]. Figure 2: Isoprenoid biosynthesis pathway. The MVA pathway (**Left**). Enzymes: Erg10, acetoacetyl-CoA thiolase; Erg13, 3-hydroxy-3-methylglutaryl-CoA synthase; Hmg1/2, 3-hydroxy-3-methylglutaryl-CoA reductase; Erg12, mevalonate kinase; Erg8, phosphomevalonate kinase; Erg19, mevalonate diphosphate decarboxylase; Idi, isopentenyl diphosphate isomerase. The MEP pathway (**Right**). Enzymes: Dxs, 1-deoxy-D-xylulose-5-phosphate synthase; Dbx, 1-deoxy-D-xylulose 5-phosphate reducoisomerase; IspD, 4-diphosphocytidyl-2-C-methyl-D-erythitol synthase; IspE, 4-diphosphocytidyl-2-C-methylerythitol kinase; IspF, 2-C-methyl-D-erythitol 2,4-cyclodextrin diphosphate synthase; IspG, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase; IspH, 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase. The synthesis of santalene and santalol requires the utilization of FPP as a central building block, serving as the fundamental precursor for all sesquiterpenes. Farnesyl diphosphate (FPP) is created through successive prenylation processes, wherein a DMAPP carbocation initially combines with one IPP unit via an electrophilic alkylation reaction and is subsequently deprotonated to yield GPP--the addition of another IPP unit to GPP results in FPP formation whereby the enzyme farnesyl diphosphate synthase catalyzes these prenylation reactions [59]. The santalene synthases accomplish the conversion of FPP into santalene [59; 60]. Multiple isoenzymes of santalene/bergamotene synthase (STS) have been characterized from _Santalum_ species [61] and other plant species like _Clausena lansium_[12; 62], as well as a camphor tree, _Cinnamoun camphora_[63].