## 3 Discussion  
In this study, _E. coli_ was engineered for the efficient conversion of MVA to (\(-\))-\(\alpha\)-bisabolol using the feedback-resistant MvaK1 and reinforcement of the lower MVA pathway. The feedback-resistant MvaK1 was firstly identified in the archaeon _M. mazei_. However, only a handful of studies have been carried out on terpenoid production in microbes. Recently, feedback-resistant MvaK1 enzymes were identified and characterized from _Methanosaeta concili_ (McMvaK) and _Methanocella paludicola_ (MpMvaK) [20]. The McMvaK and MpMvaK enzymes not only showed feedback resistance to DMAPP, IPP, GPP, and FPP, but also exhibited 4.9- and 5.5-fold higher affinity to MVA, respectively, than _Mm_MvaK1 [20]. Therefore, these MvaK1 enzymes may enable the enhancement of (\(-\))-\(\alpha\)-bisabolol production in engineered _E. coli_. To find the optimal conditions to efficiently convert MVA to (\(-\))-\(\alpha\)-bisabolol, we examined the inducer concentrations, cofactor, ATP, and reinforcement of the lower MVA pathway. Adding the inducers for overexpression of MVA or MEP pathway genes has been a controversial issue in the microbial production of terpenoids [21; 22; 23]. Lycopene production was reduced under all IPTG-induced conditions in _E. coli_ expressing the lower MVA pathway [23]. The leaky expression of all enzymes involved in the (\(-\))-\(\alpha\)-bisabolol production without IPTG addition exhibited the highest production among all tested IPTG concentrations [12]. The IPTG-induced overexpression of genes for (\(-\))-\(\alpha\)-bisabolol production can inhibit the essential cellular metabolism due to a deficiency of FPP or accumulation of toxic intermediates (IPP and HMG-CoA) of the heterologous MVA pathway [12]. In contrast, isoprene production increased as the IPTG concentration increased from 0.2 to 1.2 mM [24]. In this study, a small amount of IPTG was effective to increase (\(-\))-\(\alpha\)-bisabolol production in the engineered _E. coli_. Concerning the complex regulation of the MVA pathway, balancing the expression of multiple heterologous enzymes is crucial for the optimal production of (\(-\))-\(\alpha\)-bisabolol [10]. The availability of reducing cofactors such as NADH and NADPH strongly affects the yield and productivity of terpenoids in bacteria. The strengthening of the reducing power for the increased production of terpenoids has been attempted; modulation of glutamate dehydrogenase increased the production of \(\beta\)-carotene and lycopene through the increased supply of NADPH [25; 26; 27]. The overexpression of GAPDH of _C. acetobutylicum_ also resulted in the improvement of isoprene production [24]. Moreover, the replacement of NAD\({}^{+}\)-dependent GAPDH of _E. coli_ with the NADP\({}^{+}\)-dependent GAPDH of _C. acetobutylicum_ showed a 2.5-fold increase of lycopene productivity in the engineered _E. coli_[28]. We performed the fed-batch fermentation using the strain overexpressing _gap_C from _C. acetobutylicum_ and repressing the _gap_A gene through the CRISPR system under optimized conditions (1000 rpm and the addition of IPTG at a concentration of 0.025 mM) (Figure S2, Table 1). Contrary to the results in batch fermentations, both strains showed a negative effect on (\(-\))-\(\alpha\)-bisabolol production and had slightly reduced cell growth when compared with the strain that did not overexpress _gap_C and repress _gap_A. It appears that the overexpression of _gap_C and the repression of _gap_A were not effective when sufficient amounts of ATP and NADPH were supplied, owing to the activation of the citric acid cycle and respiration by increasing oxygen in the cells. This observation might be consistent with a previous study that lycopene production in _E. coli_ was improved by decreasing the pentose phosphate pathway flux and increasing the tricarboxylic acid (TCA) cycle flux [26]. Additionally, NADPH has been shown to inhibit 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR),which converts HMG-CoA to MVA [10].