# Advances in the Metabolic Engineering of _Escherichia coli_ for the Manufacture of Monoterpenes
## 2 Engineering Endogenous MEP Pathway in Prokaryotic Chassis
KnockS and HHCMs from _Saccharomyces cerevisiae_ & & & & & \\  & 4. EGPS from _Guzanus laevis_ & 3. PKW, PMK, and PMD from _Saccharomyces cerevisiae_ & & & & \\  & 3. EGPS from _Methus putida_ & & & & & \\  & 4. EGPS from _Guzanus laevis_ & 1. Codon optimization & & & & \\ BL21 (DE3) & 1. AtoB and IDI from _E. coli_ & 2. KnockS and HHCMs from _Saccharomyces cerevisiae_ & & & & \\  & 4. EGPS from _Guzanus laevis_ & 1. Codon optimization & & & & \\  & 1. AtoB and IDI from _E. coli_ & 2. Exogenous pathway & & & & \\  & 2. HMCS and HHCMs from _Saccharomyces cerevisiae_ & 3. Categorical evolution of FPS & & & & \\ XL-Blue & 3. MK, PMK, and PMD from _Saccharomyces cerevisiae_ & 4. Integration of seven MEV pathway genes and \(l\) in one plasmid; integration of _Sprg_ and \(l\) in another plasmid & & & \\  & 4. EGPS from _Guzanus laevis_ & 5. PS from _Pinus laevis_ & & & & \\ \hline  & & & & & & \\ Abbreviations: AtoB, acetacetyl-CoA synthase; HMGS, HMG-CoA synthase; and & & & HMGG, HMG, HMG-CoA reductase; MK, mexolonate kinase; PMK, phosphoemyloane kinase; PMD, mexolonate pyrophosphate decarboxylase; Mvzk, bifurcational acetacetyl-CoA thiolase and HHCM-CoA reductase; MvzS, HMG-CoA synthase; MvzkCl, mexolonate kinase; MvzkZ, phosphoemyloemyloemyloemyloemyloemyloemyloemyloemyloemyloemyloemyloemyloemyloemyloyloemyloyloemyloemyloemyloemyloemyloyloemyloemyloyloemyloemyloyloyloemyloyloemyloyloemyloyloyloemyloylprimarily synthesized to FPP and higher polyprenyl diphosphates rather than to the intermediate of GPP and its downstream monoterpenoids [41; 42]. Thus, introducing exogenous catalytic enzyme genes that encode GPP synthase (GPPS) and other monoterpene synthases to improve GPP production could be an appropriate solution. [PERSON] et al. tested the function of a short exogenous metabolic pathway for the biosynthesis of the simple monoterpene carvone [29]. They incorporated four enzymes, namely, GPPS, limonene synthase (LS), cytochrome P450 limonene hydroxylase (L6H), and carved dehydrogenase (CdH), into _E. coli_, which could theoretically catalyze IPP and DMAPP to carvone. However, the production of the intermediate limonene increased to nearly 5 mg/L, whereas the target product of carvone could hardly be detected. Intriguingly, feeding with exogenous limonene could push forward the carbon flux to the synthesis of carvone probably through increasing the supply of substrates. This strategy could improve the production of carvone into 0.25 mg/L. These results suggest that the endogenous supplies of the crucial precursors of IPP and DMAPP are at a relative low flux to the monoterpene synthesis. The requirement of the industrial production of monoterpens may hardly be satisfied by simply incorporating several downstream enzymes to the MEP pathway. Since the efficiency of the endogenous MEP pathway became a limiting factor, several groups attempted to engineer the MEP pathway to acquire higher titers of monoterpene. Enzymes 1-deoxy-d-xylulose-5-phosphate synthase (DXS), DXP reductoisomerase (DXR), and isopentenyl diphosphate isomerase (IDI) were demonstrated as the rate-limiting factors in the MEP pathway [43; 44]. DXS catalyzes the formation of DXP, DXR reduces DXP to 2-C-methyl-D-erythritol-4-phosphate, and IDI catalyzes the conversion of the relatively unreactive IPP to the more-reactive electrophile DMAPP. The strategies of introducing exogenous rate-limiting enzymes with high expression by codon-optimization and/or increasing the expression levels of these endogenous enzymes through integrating with strong promoters could successfully control flux from the target precursors to the subsequent desired compounds. [PERSON] et al. embedded two exogenous genes encoding GPPS and LS in _E. coli_ so that the production of limonene reaches 4.87 mg/L.