## 4 Isoprenoid Production in Methanotrophs
### Advances in Engineering Central Carbon Metabolism to Improve Isoprenoid Production in Methanotrophs  
As noted above, the MEP pathway serves as a good candidate to enhance isoprenoid production in methanotrophs by metabolic engineering. The MEP pathway uses glyceraldehyde 3-phosphate and pyruvate in the same molar ratio. However, the EMP pathway that is the main route for C1 assimilation in type I methanotrophs with 75% pyruvate produces an imbalanced ratio of G3P and pyruvate, which is unfavorable for the efficiency of the MEP pathway. Engineering the feeding module of these precursors via the Entner-Doudoroff (ED) pathway suggests the possibility to achieve the balancing of precursor pool, which subsequently improves the isoprenoid titer. The isoprene titer was increased 3.1-fold compared to the strain that used the EMP pathway only by overexpressing the ED and the pentose phosphate pathways [56]. Reconstruction of the MVA pathway in type I methanotrophs is another promising strategy for isoprenoid production if the intracellular concentration of acetyl-CoA(which is a critical branch-point metabolite in central metabolism) can be enhanced. An effective approach for metabolic engineering to improve acetyl-CoA pools in methanotrophs could increase carbon flux through the phosphoketolase (PKT) pathway (Figure 2). The overexpression of this pathway in type I methanotrophs has been successfully used to increase the yield of acetyl-CoA. High carbon flux through the PKT pathway was achieved by the overexpression of two PKT isoforms in _M. buryatense_; among them, _ptkB_ overexpression showed a two-fold increase in intracellular acetyl-CoA [57]. Therefore, the integration of PKT and MVA pathways in type I methanotrophs could improve isoprenoid production in type I methanotrophs. Furthermore, since glycogen and extracellular polymeric substance are the main carbon sink in Group I strains, the elimination of these processes could also increase the pool of secondary metabolites.  
The serine cycle is the main route for the C1 assimilation pathway in type II methanotrophs, in which formaldehyde was converted to formate and further incorporated into the serine cycle to produce acetyl-CoA. It should be noted that the production of acetyl-CoA from the serine cycle enhances the overall carbon conversion efficiency compared to RuMP-based C1 assimilation without loss of CO\({}_{2}\)[17]. Type II methanotrophs possess higher flux to acetyl-CoA production than type I methanotrophs and could be increased by eliminating polyhydroxybutyrate accumulation. Therefore, isoprenoids could also be produced by using both the heterologous expression of the MVA pathway and the endogenous MEP pathway. During initiation of isoprenoid biosynthesis via the MVA pathway, two molecules of acetyl-CoA are condensed to form acetoacetyl-CoA by an acetoacetyl-CoA thiolase. However, the heterologous expression of this enzyme has often been difficult to express in non-native hosts. Interestingly, this enzyme is also already presented in the primary metabolism of type II methanotrophs  
Figure 2: Central metabolism in methanotrophs and proposed engineering strategies for isoprenoid production. Red arrows: predicted overexpression targets to improve carbon conversion efficiency for production of isoprenoid via MEP and mexulonic acid (MVA) pathway; red X: suggested knockout targets for improving flux into isoprenoids; blue arrows: heterologous expression of MVA pathway in methanotrophs.  
via the EMC pathway. This feature could be an advantage for engineering isoprenoid biosynthesis in type II methanotrophs (Figure 2). By applying this approach, reconstruction of the MVA pathway in a model methylotroph, _Methylobacterium extonquens_ AM1, resulted in humulene concentrations of up to 1.65 g/L from methanol [58].