# Biological Properties of Sandalwood Oil and Microbial Synthesis of Its Major Sesquietrepenoids
## 4 Enzyme Engineering to Improve Catalytic Properties  
The catalytic efficiency of natural sesquiterpene synthases is generally low, affecting sesquiterpenoids' biosynthetic efficiency. For enzyme engineering, undertaking the following four areas of mutations may enhance the catalytic efficiency of sesquiterpene synthases: (1) explicitly modifying conserved structural domains, (2) aligning active site residues of sesquiterpene synthases, (3) analyzing a multitude of homologous gene sequences followed by selecting the most conserved residues as candidates for targeted mutations, and (4) combining error-prone PCR with high-throughput screening. Focusing on the C-terminus active region of \(\alpha\)-santalene synthase (CISS), the strain with a single mutation CISS\({}^{5533\AA}\) has been shown to significantly increase the production of \(\alpha\)-santalene by 1.7 folds compared to the control strain. The mutation of residue S533 to Ala resulted in the addition of two hydrogen bonds near this site (<4 A), which resulted in improving the catalytic efficiency of CISS [68]. Fusion protein will reduce the spatial distance between the substrate and the enzyme, thereby enhancing the catalytic efficiency of sesquiterpene synthases. Through expression of the fusion proteins with ERG20 and related terpene synthases, some terpenes, like (+)-Valencene, \(\alpha\)-farnesene, and patchoulol, have been synthesized efficiently in microorganisms [69; 70; 71]. While there are no reports of employing this strategy to enhance the titer of santalene, it is certainly a promising approach for increasing the catalytic efficiency of santalene synthase. QM/MM computational simulations were employed to obtain the catalytic reaction energy profiles for SaSSy and SanSyn. It was found that T318 in SaSSy and T298 in SanSyn can be involved in the deprotonation of the carbocation intermediate. F441 of SanSyn is seen as a key residue restricting the conformational dynamics of the intermediates, and thereby, the direct deprotonation by the general base T298 dominantly produces \(\alpha\)-santalene. The subsequent mutagenesis of this plastic residue leads to generating a mutant enzyme, SanSyn F441V, which can make both \(\alpha\)- and \(\beta\)-santalenes. By this method, the enzyme specificity and the plant essential oils with desirable component ratios were obtained by the combination of metabolic and enzymatic engineering [72]. Except for sesquiterpene synthases, the engineering of P450 monooxygenases is crucial for enhancing santalol biogenesis. The P450 monooxygenases form a broad and versatile family of oxidative enzymes ubiquitously found within organisms. They are critical in catalyzing most rate-limiting reactions in terpenoid biosynthetic pathways. These enzymes carry out particular oxidation reactions, utilizing their intricate catalytic mechanisms to transform various chemical substances, including drugs, toxins, and endogenous molecules. The P450 enzymes are typically involved in single electron transfer processes, which activate oxygen to generate intermediates, subsequently oxidizing the substrates. The P450 core structure consists of a heme-iron reactive center with the catalytic site around the heme-containing comparatively small peptide regions (termed substrate recognition sites, SRS) involved in substrate binding and catalysis. These SRS show high amino acid sequence variation, underlying the ability of different P450s to bind diverse substrate types [73]. The N-terminus membrane binding region of P450 is also essential for its expression and solubility. Furthermore, the co-expression of a plant CPR, the reductive partner of P450, is required to obtain the high plant P450 activity in heterologous hosts [74]. The low heterologous expression, poor stability, and low catalytic activity of P450 represent the bottlenecks of santalol heterologous biosynthesis. However, there has yet to be a report about the engineering modification of P450 for the formation of santalol.