# Advances in the Metabolic Engineering of _Escherichia coli_ for the Manufacture of Monoterpenes
## 3 Introduction of Heterologous MEV Pathway  
Bypassing the inherently metabolic synthesis pathway through replacing it with a heterologous mevalonate-dependent pathway provides a pioneering strategy for the production of valuable terpenoids [46]. For example, [PERSON]'s group introduced a heterologous MEV pathway into _E. coli_ to increase amorphadiene titer greater than 100 mg/L [47]. This mechanism might be the overproduction of universal precursors IPP and DMAPP by the heterologous expression of MEV pathway enzymes with terpene synthases, which could enhance the conversion efficiency of IPP and DMAPP to relevant terpenoids. Similar strategies were inspired to increase the titers and yields of various monoterpens. For instance, [PERSON] et al. engineered _E. coli_ by introducing all seven enzyme-encoding genes involved in the MEV pathway and downstream limonene and perillyl alcohol (POH) synthesis-dependent genes encoding GPPS, LS, and cytochrome P450 [32]. Considering that the dispersion of the seven genes of the MEV pathway into multiple plasmids could aggravate the metabolic burden and hinder the target product synthesis in the host cell due to the raw materials and energy consumption [48; 49], the authors integrated the seven genes into one plasmid and generated various versions of plasmid constructs for further study. The appropriately modified metabolic route increased the yield of limonene to 435 mg/L during 72 h of incubation with supplement of 1% glucose in the culture medium. Thus, the high titer of limonene could overcome its uptake and trafficking restrictions, which might promote the conversion efficiency of limonene to the target monoterpene such as carvone. Accordingly, the strain harboring the reasonably combinatory plasmids was induced to produce its derivative POH to about 34 mg/L. For \(\alpha\)-pinene production, [PERSON] et al. embedded the heterologous hybrid MEV pathway and the co-expressed GPPS and \(\alpha\)-pinene synthase (PS).  
The final biosynthesis production accumulated up to 5.44 mg/L and 0.97 g/L under the culture conditions of flask and fed-batch fermentation, respectively [50]. Another intriguing work indicated that the incorporation of GPPS and \(\beta\)-phellandrene synthase (PHLS) in _E. coli_ could not result in the measurable yield of \(\beta\)-phellandrene. However, after introducing the MEV pathway in collaboration with the GPPS and the PHLS, the output of \(\beta\)-phellandrene reached 11 mg/g\({}_{\mathrm{dcw}}\) after over 20 h of incubation. This titer could be further improved to 25 mg/g\({}_{\mathrm{dcw}}\) by optimizing LB broth with 1% glucose supplement and then extending the incubation time to over 72 h [33]. Notably, when the endogenous MEP pathway and the exogenous MEV pathway were used individually, the production of sabinene generated from integrated GPPS and sabinene synthase in the MEV pathway was 20-fold higher than that in the MEP pathway. The final production via a series optimization for culture condition and inducer concentration achieved a maximum titer of 82.18 mg/L under shake-flask culture and 2.65 g/L under fed-batch culture [36]. However, the method of heterologous MEV pathway introduction is not always available for different monoterpene biosynthesis to reach an industrial grade, which is probably due to restrictions of the rate-determining step and the imbalance of the metabolic flux. These bottlenecks may result in the accumulation of some toxic intermediates and the limitation of the downstream essential products in the intracellular space.