# 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  
It is necessary to modify poorly active monoterpene/sesquiterpene synthases through enzyme engineering. Common enzyme engineering methods include truncation, fusion, site-specific mutagenesis and directed evolution (Figure 2B) [94]. Plant-derived monoterpene synthases are typically localized to specific organelles in plants, such as mitochondria, chloroplasts or the endoplasmic reticulum. These enzymes often contain an \(N\)-terminal plastid targeting sequence (PTS) in their sequence [14]. When plant-derived terpene synthases are introduced into microbial cells, the cells may not recognize and cleave the signal peptide sequence, resulting in inefficient expression of the terpene synthases. Truncating the \(N\)-terminus of the PTS is an effective method to increase the activity of monoterpene synthases. For example, the strain expressing truncated linalool synthases showed an 8-fold increase in linalool production compared to the strain expressing with an untruncated enzyme [92]. For the synthesis of myrrene, the truncation of seven myrrene synthases from different sources revealed that trCsMS from _Cannabis sativa_ exhibited the best activity [70]. Moreover, the activity of the same monoterpene synthase can be affected by different truncation positions. For instance, [PERSON] et al. truncated the linalool synthase at two different sites and found that the linalool synthase from _Mentha citrata_ with RR site truncation (t67-McLIS) showed the best activity, increasing the linalool production by 17 times compared to that of the untruncated synthase [95]. [PERSON] et al. performed truncation at four different sites (S14, L28, S43 and S52) of the geraniol synthase (CrGES) derived from _Catharanthus roseus_ and identified that the S43-truncated CrGES exhibited the highest activity, with geraniol production reaching 191.61 mg/L, 4.45 times higher than that of the full-length CrGES [96]. Fusion expression of terpene synthases can also effectively enhance their catalytic activity. Fusion expression with short peptide tags is an effective method, as the addition of short peptide tags does not cause steric hindrance and can increase the translation efficiency of the recombinant proteins. [PERSON] et al. fused the linalool synthase with MBP, NusA, GST and the short peptide tag CmR29 and found that only the expression of the CmR29*bLIIS fusion protein increased the linalool production, confirming the effect of short peptide tags in enhancing terpene synthase activity [97]. Subsequently, they also fused the linalool synthase with the CmR29 tag, resulting in a linalool titer of 1342.6 mg/L [72]. [PERSON] et al. fused three variants of CMR29 to the \(N\)-terminus of the santalene synthase and obtained the highest santalene production of 1078.8 mg/L, which is 131% higher than that of the strain without the tagged synthase [98]. Additionally, the fusion of terpene synthases with GPP/FPP synthases can effectively increase the production of monoterpenes/sesquiterpenes. [PERSON] et al. fused the sabinene synthase (SpSabS1) from _Salvia pomifera_ with ERG20, resulting in a 3.5-fold increase in sabinene production [99]. [PERSON] et al. fused the patchoulol synthase with FPP synthase and found that a short linker was more effective in enhancing patchoulol synthase activity [100]. [PERSON] et al. found that fusion expression of the phellandrene synthase and GPP synthase with a tandem Gly-Ser-Gly (GSG) linker could promote phellandrene production, achieving the highest phellandrene production of 32 mg/L [101]. Recently, [PERSON] et al. fused the nerolidol synthase with FPP synthase using four different linkers, leading to a 110-fold increase in nerolidol shake-flask production, reaching 4.2 g/L [74]. Moreover, synthetic enzymes can also be co-localized on an artificially created protein scaffold for expression. The synthetic protein scaffold can help intermediates in the metabolic pathway transfer directly from one enzyme to the next, enhancing synthesis efficiency. [PERSON] et al.