# Advances in the Metabolic Engineering of _Escherichia coli_ for the Manufacture of Monoterpenes
## 4 Optimization of the Expression and Function of the Rate-Limiting Enzymes  
A common method to break the restrictions of the rate-determining step is to increase the expression level of rate-limiting enzymes in prokaryotic hosts. Three strategies are available: (1) insertion of stronger promoters into the operons directing the enzyme gene expression; (2) codon optimization of enzyme-coding regions; and (3) screening of enzymes from different species for a higher compatible and efficient homolog [51; 52]. For the integration of MEV pathway and monoterpene synthases, a series of strong promoters, including T7, lacUV5, and trc, was utilized [32; 33]. In another way, the codon optimization scheme is also essential for the heterologous metabolic system due to relatively diminished expression efficiency by biased codon usage [53]. Moreover, for the enzyme homolog screening, GPPS is considered a crucial candidate, which could be classified as homomeric and heteromeric isoforms [54; 55]. The heteromeric GPPS extracted from _Mentha piperita_ is composed of two subunits with different sizes, which shows no single catalytic activity. This heterodimer could transform IPP and DMAPP to C\({}_{10}\) GPP and C\({}_{20}\) GGPP [56]. The homomeric GPPS from _Arabidopsis thaliana_ is a polyphenyl pyrophosphate synthase that synthesizes multiple terpen ranging from C\({}_{25}\) to C\({}_{45}\), while another type from conifers, such as _Picea abies_ and _Abies grandis_, yields only GPP [41; 57; 58]. Because the GPP specific-producing manner can avoid or suppress the byproduct generation and effectively control the metabolic flux into the monoter, the fully characterized GPPSs from _P. abies_ and _A. grandis_ were confirmed the better synthetases for the monoterpene production in host microbes, which need further codon optimization and truncation of tail sequences encoding plastid signal peptide for prokaryotic cell integration.  
Another striking method for the functional improvement of the enzymes is directed evolution, which relies on random mutagenesis and high-throughput screening [59]. It confers the enzymes with an unnatural powerful catalytic efficiency and effectively overcomes the metabolic flux nodes [60; 61]. Notably, the monoterpene synthase-mediated reactions are widely considered as the rate-limiting step in production. The improved activity and stability of enzymes could benefit to compete for the essential intermediate GPP from FPPS, which will lead the carbon flow to the final monoter. For instance, [PERSON]'s group isolated a PS variant that outperforms the wild-type through the directed enzyme evolution. After co-expression of this variant with IDI, GPPS, and the MEV pathway, pineine productivity could reach 140 mg/L in flask culture [40]. Technically, the visualization of substrate consumption and the diversity of alternative variants could facilitate the establishment of the screening system for the directed evolution of enzymes [62; 63]. Thus, this approach could rapidly select perfect mutants for advanced cellular performance, which is suggested to be suitable for many other enzymes in the metabolic route [59].