# Biosynthesis Progress of High-Energy-Density Liquid Fuels Derived from Terpenes
## 4 Strategies for Constructing Efficient Microbial Cell Factories for Terepenes
### Optimizing Metabolic Pathways  
#### 4.2.1 Optimization of MEP and MVA Pathways  
In _E. coli_, the precursors IPP and DMAPP produced through the MEP pathway are in low amounts, resulting in the limited synthesis of terpen. For example, when introducing farnesene synthase into _E. coli_ BL21(DE3) to synthesize farnesene with the MEP pathway, the production of farnesene is only 0.325 mg/L [109]. Similarly, when expressing the sesquiterpene synthases EIZS, PentS and MrTPS2 in _E. coli_ DH1, the production values of epi-isozizaene, pentalene and \(\alpha\)-isocometene are only 0.54 mg/L, 0.19 mg/L and 0.01 mg/L, respectively [38]. Increasing the precursor metabolic flux is an effective strategy to improve the production of terpen (Figure 2B). The rate-limiting steps in the MEP pathway are not well understood, and enzymes such as Dxs, IspD and IspF in the pathway are subject to feedback inhibition [110; 111]. Overexpression of these enzymes leads to the accumulation of the intermediate metabolite MECPP (2-C-methyl-D-erythritol-2,4-cyclodiphosphate), which limits the enhancement of terpene production via modification of the MEP pathway [112]. Researchers have shown that introducing the exogenous MVA pathway for increasing the precursor supply is more beneficial than modifying the MEP pathway in _E. coli_[113]. The MVA pathway genes are a requirement, and are commonly derived from _Enterococcus faecalis_, _Saccharomyces cerevisiae_, _Streptomyces_ sp. CL190, etc. [114]. For instance, [PERSON] et al. constructed a plasmid containing all genes from the MVA pathway derived from _S. cerevisiae_ and achieved a limonene titer of 400 mg/L in shake-flask fermentation using glucose as the carbon source [115]. Subsequently, the limonene production was further enhanced to 3.1 g/L through the optimization of fermentation [45]. [PERSON] et al. separated the _atobB_, _HMGS_, _HMGR_, _MK_, _PMK_, _PMD_ and _idi_ into two expression cassettes and introduced the resulting plasmid into _E. coli_ MG1655, and the production of pinene increased to 104.6 mg/L, which was 13.1 times that of the initial strain [116]. In addition, the production of sesquiterpenes, including caryophyllene, \(\beta\)-eudesmol, farnesol and protoilludane, was also increased through the expression of the exogenous MVA pathway, codon optimization, etc. [117; 118; 119; 120]. Moreover, the synthesis of farnesene (1.1 g/L) expressing the exogenous MVA pathway, FPP synthase and farnesene synthase in _E. coli_ was 2000 times than that of strains expressing only farnesene synthase [109]. Typically, exogenous pathway genes are constructed into plasmids and transformed into _E. coli_ for the synthesis of targets. However, the genetic instability of plasmids and the metabolic burden imposed by antibiotic expression affect the high-density growth of bacterial cells and product generation. Chromosomal integration effectively addresses these issues [121; 122]. For example, in order to overcome the disadvantages of plasmid expression during \(\beta\)-carotene production, _HMG1-ERG12_ and _mux5-muxA-muxD1_ were integrated into the _E. coli_ chromosome at the _p/IB_ and _frdB_ sites. By further regulating the expression of _atobB_, _mux_ and _HMG1_, the titer of \(\beta\)-carotene increased 1.51-fold without affecting the cell growth [123]. However, the gene copy number for chromosomal integration is lower than that of plasmid expression, resulting in lower expression levels. To address this issue, exogenous genes are often expressed under the control of strong promoters (e.g., T7), integrated into multiple copy loci in the genome or expressed by increasing the copy numbers of chromosomal integration [122]. [PERSON] et al. used CRISPR-Cas9 to construct the MVA pathway and squalene synthase into three expression cassettes, including T (pKICO-CmoxB-PgadE-MevT), M (pKICO-Kan-lacZ-Ptr-MevB) and B (pKICO-Kan-rbsAR-LacI-Ptr-BS-idi-ispA), and integrated the three cassettes into the _rbsAR_, _lacZ_ and _porxB_ loci in _E. coli_, respectively.