## 2 Results
### Effect of Aeration on (\(-\))-\(\alpha\)-Bisabolol Fermentation  
We performed a fed-batch fermentation to produce the (\(-\))-\(\alpha\)-bisabolol in _E. coli_ DH5\(\alpha\) containing both pTSN-Bisa-Mm and pSSN12 Didi-MrBBS-IspA plasmids. The yield of (\(-\))-\(\alpha\)-bisabolol was improved by 16% in 46 h compared to when the lower MVA pathway was not additionally overexpressed. However, the MVA still accumulated from the beginning of fermentation and reached 10.1 g/L in 68 h at 280 rpm despite reinforcing the lower MVA pathway (Figure 6A). It seems that there are other bottlenecks when MVA is converted to (\(-\))-\(\alpha\)-bisabolol through the lower MVA pathway. The synthetic MVA pathway requires 3 moles of ATP to convert MVA to (\(-\))-\(\alpha\)-bisabolol (Figure 1) and competes for the ATP with other essential cellular reactions involved in cell growth [18]. Because ATPs are efficiently generated under aerobic conditions using NADHs in oxidative phosphorylation [19], aeration effects were examined by controlling the agitation speed in fed-batch fermentation.  
Figure 5: Overexpression of _gapC_ gene encoding NADP\({}^{+}\)-dependent GAPDH from _C. acetobutylcium_ and repression of _gapA_ gene coding for endogenous NAD\({}^{+}\)-dependent GAPDH from _E. coli_ using the CRISPR system. (**A**) Plasmid constructs for expressing genes of the entire (\(-\))-\(\alpha\)-bisabolol biosynthetic pathway introducing the _gapC_ gene downstream of the _ispA_ gene (pTSN-Bisa-Mm-GapC) and for expressing inactivated Cas9 (dCas9) and sgRNA targeting the _gapA_ gene (pdCas9-sgRNA-GapA). (**B**) Comparison of (\(-\))-\(\alpha\)-bisabolol concentrations produced by the _E. coli_ DH5\(\alpha\) harboring pTSN-Bisa-Mm, pTSN-Bisa-Mm-GapC, or both pTSN-Bisa-Mm-GapC and pCas9-sgRNA-GapA. (**C**) Comparison of cell growth of the strains. Cells were grown in TB medium containing 10 g/L glycerol and 20% (v/v) of _n_-dodecane in the presence of different IPTG concentrations (0, 0.025 and 0.1 mM) at 30 \({}^{\circ}\)C for 72 h. The error bars represent the standard deviation of the concentrations of (\(-\))-\(\alpha\)-bisabolol and OD\({}_{600}\) from three biological replicates.  
In fed-batch fermentation at an agitation speed of 1000 rpm, the _E. coli_ strain harboring pTSN-Bisa-Mm and pSSN12 Didi-MrBBS-IspA rapidly grew and reached the maximum cell growth within 23 h showing a 1.8-fold improved cell growth relative to those at 280 rpm. As expected, MVA accumulation was considerably reduced, but (-)-\(\alpha\)-bisabolol production was not significantly improved (Figure 6B). It seems that acetyl-CoA was utilized for cell growth and other metabolism,  
Figure 6: Fed-batch fermentation of the _E. coli_ DH5\(\alpha\) harboring both pTSN-Bisa-Mm and pSSN12 Didi-MrBBS-IspA plasmids. The fed-batch fermentation was performed in TB medium and 20% (v/v) of _n_-dodecane using two-phase culture at 30 \({}^{\circ}\)C and pH 7.0. After depletion of glycerol initially added, glycerol was fed intermittently into the bioreactor during fermentation. An agitation speed of 280 rpm (**A**) or 1000 rpm (**B,C**) and an aeration rate of 1 vvm were maintained throughout the cultivation. After 6 h cultivation, 0.025 mM of IPTG was added to the bioreactor (**C**).  
or metabolites downstream of MVA, accumulated. To overcome this problem, we sought to enhance the overall (\(-\))-\(\alpha\)-bisabolol flux through overexpressing all genes in the (\(-\))-\(\alpha\)-bisabolol biosynthetic pathway by the addition of IPTG. We carried out the fed-batch fermentation at 1000 rpm and supplied IPTG at a concentration of 0.025 mM after 6 h of incubation. Similar to the culture in the absence of IPTG, cells rapidly grew for 22 h, but MVA did not accumulate during fermentation, supporting the lower pathway was intensified by IPTG addition. The yield and productivity of (\(-\))-\(\alpha\)-bisabolol were improved by 40% and 56%, respectively, as compared to the absence of IPTG, and the final titer of (\(-\))-\(\alpha\)-bisabolol reached 8.5 g/L (Figure 6C).