# Advances in the Metabolic Engineering of _Escherichia coli_ for the Manufacture of Monoterpenes
## 1 Introduction  
Terpenoids are widely distributed natural compounds that are extracted from plants, algae, mosses, and even insects and microbes. Terpenoids are composed of isoprene five-carbon units (C5) as the basic skeleton in accordance with the biogenic isoprene rule and then further classified as monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenoids (C30), tetraterpenes (C40), and polyterpenes. These numerous compounds are normally used as medicines, insecticides, and fragrances [1]. For example, artemisinin is an endoperoxidase sesquiterpene lactone isolated from _Artemisia annua Linn_ and used as an anti-malarial drug [2]; paclitaxel is a cyclic diterpene hydrocarbon derived from the pacific yew and broadly applied in clinic as an anticancer drug [3; 4]; squalene is the precursor of triterpenoids and is used as a pharmaceutical intermediate and bactericide [5; 6]. All isoprenoids are synthesized from co-precursor isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), and this process is catalyzed by a series of corresponding isoprenoid synthases. IPP and DMAPP are subsequently transformed to geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), or geranylgeranyl pyrophosphate (GGPP), which are the precursors of monoterpenes, sesquiterpenes, and diterpenes, respectively [7]. Two major pathways are involved in the natural synthesis of isoprenoid precursors IPP and DMAPP: the methylerythrid-4-phosphate-dependent pathway (MEP pathway), also termed as deoxyxylulose phosphate pathway (DXP pathway) and the mevalonate-dependent isoprenoid pathway (MEV pathway) [8; 9]. Almost all eukaryotes and archaea use the MEV pathway,whereas most prokaryotes take advantage of the MEP pathway. Plants can utilize both biosynthetic pathways [10; 11; 12] (Figure 1). Monoterpenes, the members with the smallest molecular weight in terpenoids, are gaining significant attention because of their various applications in medicines, biofuels, and agriculture in addition to the traditional use of essential oils and flavor production. Monoterpenes have been gradually recognized as essential medicines and prophylactic formulations because of their characteristics that can be easily absorbed by the body and transferred into blood and their ability to treat severe chronic diseases, including cancer [13]. Limonene, which is used to produce fragrance, flavor, and medicinal products, is a compound generally recognized as safe because of its earth-friendly cleaning performance [14; 15]. Geraniol is often used in the production of perfumes and cosmetics, and can also be used as a clinical anticancer drug against pancreatic, colon, and other cancers [16; 17; 18]. Meanwhile, some monoterpenes and their derivatives, such as \(\alpha\)-pinene, camphene, and limonene, have high calorific value of combustion and low freezing point; these advantages make them a favorable choice for next-generation clean jet-b biofuels to replace the traditional jet fuels such as JP-10 and RJ-5 [19; 20]. Moreover, some monoterpenes, such as carvacrol, p-cymene, and gamma-terpinene, are toxic to microbes and insects, and are thus often used as antibiotics and insecticides [21; 22]. Figure 1: Monoterpene synthesis pathway in engineered prokaryotic host. GA3P, D-glyceraldehyde 3-phosphate; CoA, coenzyme A; MEP, methylerythtitol 4-phosphate; DXS, 1-deoxyD-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5 phosphate reductoisomerase; AtoB, acetoacetyl-CoA synthase; HMGS, 3-hydroxy3-methylglutaryl-CoA synthase; HMGR, 3-hydroxy-3 methylglutaryl-CoA reductase; IDI, isopentenyl diphosphate isomerase; DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate. In the past, monoterepenes were mainly obtained from plant biomass, but this traditional production method restricts their wide applications because of its low yields, high costs, long reaction cycles and difficult purification [23].