# 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  
Metabolic engineering strategies have shown promise in transforming microorganisms such as yeast and bacteria into effective cell factories for enhancing the production of certain chemical substances. As stated earlier, the availability of sandalwood oil intensely depends on extracting it from the heartwood part of sandalwood. The sustainability of natural sources of sandalwood oil, such as _Santalum_ species, is uncertain due to overexploitation, long growth time to maturity, and climate changes [78]. Records show that the annual consumption of Indian sandalwood is approximately 4000 tonnes. According to 2004 records, oil production has decreased drastically to around 70 tonnes compared to the last six decades, when the output of Indian sandalwood oil was nearly 180-200 tonnes, sufficient to meet consumer demand. This drastic fall in sandalwood oil production is due to a shortage of sandalwood trees [79]. Overcoming these limitations necessitates adopting a sustainable approach for the large-scale production of santalene and santalol to meet commercial demands [16].  
_S. cerevisiae_ is among the numerous microorganisms used for centuries to synthesize valuable biomolecules. It is a single-celled eukaryotic microorganism belonging to the fungi kingdom and is the most extensively studied eukaryotic microbe, providing insight into the biology of eukaryotic cells [80]. Due to its non-pathogenic characteristics, _S. cerevisiae_ is considered a GRAS organism (Generally Recognized as Safe). _S. cerevisiae_ is a valuable asset in biotechnology, offering many possibilities for innovative applications and advancements. It is known for its ability to utilize various carbon sources, ease manipulating sterol pathways by introducing exogenous genes such as TPSs [81; 82], robustness, and adaptability to harsh conditions [13]. This makes _S. cerevisiae_ an indispensable tool in the biotechnological production of terpenoids.  
In _S. cerevisiae_, the IPP and DAMPP are generated through the MVA pathway. In the first step, acetyl-CoA molecules are transformed into acetoacetyl-CoA catalyzed by acetoacetyl-CoA thiolase enzyme (ERG10). The subsequent condensation of acetoacetyl-CoA with another acetyl-CoA results in the formation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) catalyzed by HMG-CoA synthase (ERG13). Following this, HMG-CoA's thioester is converted into alcohol by the HMG-CoA reductase enzyme, producing mevalonate. This set of reactions involves two consecutive reduction reaction mechanisms, each demanding one NADPH H\({}^{+}\) molecule: the first for the conversion of the thio-esterified carboxyl group into an aldehyde and the second for the transformation of the aldehyde into alcohol [83; 84; 85]. In _S. cerevisiae_, two isoenzymes exist: Hmg1 and Hmg2. These isoenzymes exhibit HMG-CoA reductase activity, with Hmg1p noted for their prominent role in yeast enzyme activity [86]. Both Hmg1 and Hmg2 are located in the endoplasmic reticulum membrane. Then, mevalonate undergoes two consecutive phosphorylation reactions catalyzed by ERG12 and ERG8 to yield mevalonate phosphate and mevalonate diphosphates, respectively, whereby the second phosphorylation reaction is reversible. The subsequent response involves the decarboxylation of mevalonate diphosphate to yield IPP, a reaction catalyzed by ERG19, which is ATP-dependent. Lastly, DMAPP is generated through an isomerization reaction of IPP by isopentenyl diphosphate isomerase (IDI1) [87]. The santalenes and santales can be obtained by reconstructing the biosynthetic pathway of santalenes and santalos in _S. cerevisiae_ through the introduction of exogenous genes such as STSs, which converts FPP into santalenes and CYPs, alongside its redox partner cytochrome P450 reductases (CPRs), which oxidizes santalenes into santalos (Figure 3). The following metabolic engineering strategies have been employed to increase the yield of santalene and santalol in _S. cerevisiae_.