## 4 Isoprenoid Production in Methanotrophs  
### Isoprenoid Biosynthesis Via MEP Pathway  
The basic isoprenoid building blocks isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are synthesized through one of two distinct biosynthetic routes--the mevalonic acid (MVA) pathway or the 2-C-methyl-b-erythnitol 4-phosphate (MEP) pathway. Over the last few decades, the MVA pathway was considered to be the unique pathway for IPP and DMAPP biosynthesis [33]. However, in the early 1990s, an alternative pathway named the MEP pathway was discovered in bacteria for the biosynthesis of IPP and DMAPP (Figure 1). While archaea and most eukaryotes use the MVA pathway for IPP and DMAPP synthesis, the bacteria (including key pathogens such as all Gram-negative bacteria and mycobacteria) use the MEP pathway [34]. The MEP pathway consists of seven enzymatic steps which initiate with the condensation of pyruvate and p-glyceraldehyde 3-phosphate (G3P) to form 1-deoxy-p-xylulose 5-phosphate (DXP) by 1-deoxy-p-xylulose-5-phosphate synthase (DXS). Next, DXP is converted to MEP by 1-deoxy-p-xylulose-5-phosphate reductoisomerase (DXR) and is subsequently converted to methylerythnitol 2,4-cyclodiphosphate (MEcDP) through three consecutive enzymatic steps, cytidylation (CTP dependent), phosphorylation (ATP-dependent), and cyclization encoded by _ispD_, _ispE_, and _ispF_. In the next step, MEcDP is converted to hydroxymethylbutenyl diphosphate (HMBDP) catalyzed by HMBDP synthase (HDS) encoded by the _ispG_ gene. In the final step, HMDBP is reduced to IPP and DMAPP by the action of the _ispH_ gene product, HMBDP reductase (HDR). IPP and DMAPP are isomerized by isopentenyl diphosphate isomerase (IDI) [35]. The regulatory network of the MEP pathway consists of different regulatory mechanisms involving intermediate metabolites and enzymes. Methylerythnitol cyclodiphosphate (MEcDP), the fifth intermediate of this pathway, was recently reported as a key metabolite and may play a critical role in MEP pathway regulation. MEcDP may connect the MEP pathway with other cellular metabolism in making precursors for isoprenoids [35]. An efficient fermentation-based process for isoprenoid production has been achieved in engineered _E. coli_ by the heterologous expression of the MVA pathway or upregulation of the native MEP pathway. Among them, the expression of the heterologous MVA pathway is the superior route for enhancing isoprenoid production compared to the MEP pathway. However, the MEP pathway possesses many advantages for isoprenoid production, including higher theoretical yield and better balance than that of the MVA pathway [36]. Specifically, the theoretical maximum IPP yield on glucose via the MEP pathway is 0.83 C-mol IPP/C-mol glucose and is higher than that via the MVA pathway, which is 0.56 C-mol IPP/C-mol glucose IPP [37]. Methanotrophs currently play an important role in a wide range of biotechnological applications with their ability to utilize single carbon (C1) feedstock, i.e., methane and methanol, to produce various high-value compounds. Our genome analysis indicated the existence of a set of genes encoding the methylerythnitol 4-phosphate (MEP) pathway in both type I and type II methanotrophs [24] (Table 1). Interestingly, many strains belong to type I methanotrophs such as _Methylomonas_ sp. DH-1, _M. alcaliphilum_ 20Z, and _M. buryatense_ 5 GB1, and possess two genes encoding DXS, which catalyzes the first step of the MEP pathway. On the other hand, some strains belonging to type II methanotrophs such as _M. trichosporium_ OB3b consisted of two copies of DXR, which converted DXP to MEP in the second step of MEP pathway (Table 1). In contrast to _E. coli_ and _Bacillus subtilis_, IDI, which isomerized IPP and DMAPP in the last step of MEP, did not exist in either type I or type II methanotrophs. These results suggest that the regulatory mechanism of the MEP pathway in methanotrophs might be different compared to other microbes, which need to be elucidated in further studies.