# Advances in the Metabolic Engineering of _Escherichia coli_ for the Manufacture of Monoterpenes
## 7 Conclusions  
The metabolic engineering of monoterene production has already achieved substantial progress in recent years. In early studies, the native MEP pathway as a primary regulatory objective acquired considerable attention. Most studies focused on the overexpression of key enzymes in this pathway, which limit productivity. Unfortunately, the implementations of such strategy could not achieve the expected effect for the industrial production of monoterenes possibly because of the intrinsic barrier of the MEP pathway for supplying IPP and DMAPP, two essential universal monoterene-building components. The introduction of a stronger heterologous MEV pathway partially compensated for this shortage. This strategy was subsequently designed to combine with a series of optimization approaches, such as functional improvement of the rate-limiting enzymes, control of the metabolic flux, and increase of the host's tolerance to the product toxicity as discussed above, for further productivity improvement. In general, most monoterene biosyntheses remain far from the industrialization by now. To address this problem, systematic analysis of the complicated metabolic system should be considered in future studies for monoterene manufacturing. One notable study designed a computational tool named principal component analysis of proteomics (PCAP), for the multi-dimensional engineering of global metabolic pathways. This rational mathematical tool interrogated the data of the targeted proteomics and products based on principal component analysis, which could help researchers to modify the expression of some specific enzymes, balance the metabolic pathways, and predict product yields. Thus, the overall strategies for engineering the higher-efficient monoterene-producing chassis could be rationally designed [90]. Future studies should also consider the systematical integration of multiple approaches demonstrated to be effective in the prokaryotic chassis. An intriguing research took advantage of a multi-level analytical method to define and assess four engineering strategies for fermentative limonene production in _E. coli:_ (1) Construction of a metabolic route by transformation of recombinant plasmids harboring heterologous MEV pathway enzymes and the subsequent synthases into the host strains; (2) Determination of the enzyme activity for choosing an appropriate GPPS-exploited cell-free system with the functional proteins extracted from the induced host and then adding extra substrates to the system; (3) Selection of the most suitable host by analyzing the physiological properties of different _E. coli_ strains after introduction of the MEV pathway and the downstream steps; (4) Utilization of two liquid-phase carbon source fed-batch fermentation with glucose and glycerol, as well as adding a non-toxic organic solvent for the in situ extraction of monoterenes [35]. This attempt of the integration scheme from genetic modification to process optimization surprisingly increased target productivity. Further studies are necessary to find other complementary advantages of various metabolic engineering approaches and systematically elongate the pipelines of engineering for monoterene biomanufacturing. Other interesting opportunities for future studies of monoterene biomanufacturing will rely on the rapid development of the synthetic biology, which could interrogate the organizational principles of metabolic systems and easily rewire the prokaryotic cells into highly efficient cell factories. The growing strategies generated from this field, such as artificial life creation, gene circuit reconstruction, and metabolism redirection, would open new opportunities not only for the synthesis of monoterenes but also for many other valuable important chemicals. Their applications in the construction and optimization of microbial cell factories will inevitably shorten the distance from laboratory trials into industrial production.