# Biosynthesis Progress of High-Energy-Density Liquid Fuels Derived from Terpenes
## 4 Strategies for Constructing Efficient Microbial Cell Factories for Terepenes
### Terpene Synthase Mining and Engineering
#### 4.1.2 Engineering of Terpene Synthases
established a terpene synthase activity screening system based on the recognition of affibodies to their anti-idiotypic partners and co-localized the FPP synthase and the farresene synthase on this scaffold in yeast. By adjusting the ratio of the protein scaffold and the enzyme, they increased farresene production by 135% [102]. Based on the crystal structure of terpene synthases or structural computational models, the rational or semi-rational design of site-directed mutagenesis is also an effective method to enhance their catalytic efficiency. Residues surrounding the active site and substratebinding site are generally considered to be closely related to the catalytic efficiency and catalytic specificity of the enzyme [103]. [PERSON] et al. performed a homology modeling analysis of the germacrene A synthase (AvGAS) based on the structure of the 1,8-cineole synthase (PDB ID: 5 NX6) and selected 16 specific residues for mutagenesis based on the combination of the structural analysis and the homology sequence analysis. They found that the mutations F23W and F23V significantly increased germacrene A production, by 35.2% and 21.8%, respectively [104]. [PERSON] et al. established the structure model of the viridiflorol synthase based on the structure of the epi-isozizaene synthase (PDB ID: 4 ltz) and 15 highly similar synthase sequences. They designed a small library targeting 16 sites and selected 15 mutations based on the phylogenetic tree. Unfortunately, these mutations led to a decrease in the target product. Subsequently, by randomly mutating 400 clones, they found that the combined V314Y and G227C mutations increased viridiflorol production by 38% [61]. Additionally, site-specific mutagenesis of enzymes can also change the type of catalyzed product. For example, [PERSON] et al. showed the plasticity of the sesquiterpene synthase and the capacity to interconvert it into novel terpene synthases by engineering the plasticity residues [105]. [PERSON] et al. used AlphaFold2 to predict the structure of the santalene synthase (SanSyn) and found that the F441 residue of SanSyn is a key residue that limits the conformation dynamics of the intermediate. After site-directed mutagenesis of F441 to F441V, the SanSyn was able to simultaneously produce \(\alpha\)- and \(\beta\)-santalene [106]. Moreover, [PERSON] et al. showed that engineering the active-site residues responsible for product specificity could convert cineole synthase to other monoterenes synthases, as well as sesquiterpene synthases [107]. In fact, site-directed mutagenesis of terpene synthases largely depends on enzyme structure information and the enzyme catalytic mechanism. The results of site-directed mutagenesis often cannot meet the expectations. The directed evolution of enzymes does not depend on the identification of enzyme structure and is also an effective method to enhance catalytic efficiency. However, enzyme-directed evolution will create a large library of enzyme mutations, and establishing an effective screening system is an essential requirement. [PERSON] et al. established a terpene synthase screening system based on the competition of the terpene synthase and the carotenoid synthase for their common substrates. Based on this system, they performed random mutagenesis and screening of the catalytic domains of phellandrene synthase, resulting in a 60% increase in phellandrene production in _E. coli_[46; 108]. Recently, our research group achieved the directed evolution of the myrcene synthase by establishing a novel myrcene whole-cell biosensor. As a result, we obtained eight improved myrcene synthase mutants, with the highest one displaying a 1.47-fold increase in catalytic efficiency compared to the parent strain and reaching a myrcene titer of 510.38 mg/L [70].