## 4 Materials and Methods
### Plasmid Construction  
The plasmids and primers used in this study are listed in Table 2 and Table S1, respectively. Standard molecular biological techniques including genomic DNA preparation, restriction digestions of DNA, plasmid transformation were performed as previously described [29]. T4 DNA ligase, and all restriction enzymes were obtained from New England Biolabs (NEB, Ipswich, MA, USA). Polymerase chain reaction (PCR) was carried out following the manufacturer's protocols with a high fidelity KOD-Plus-Neo polymerase (Toyobo, Osaka, Japan). Kits for plasmid preparation and gel extraction \begin{table}
\begin{tabular}{l c c c c c} \hline \hline \multirow{2}{*}{**Plasimids**} & **Agitation** & **IPTG** & **Final Titer** & **Initial Yield * & **Initial Productivity * \\  & **(rpm)** & **(mM)** & **(g/L)** & **(g/g)** & **(g/L h)** \\ \hline pTSN-Bisa-Mm & 280 & 0 & 8.2 & 0.08 & 0.06 \\ \hline pTSN-Bisa-Mm & 280 & 0 & 7.5 & 0.10 & 0.07 \\ pSSN12 Didi-MrBBS-IspA & 1000 & 0 & 7.0 & 0.07 & 0.09 \\  & 1000 & 0.025 & 8.5 & 0.11 & 0.12 \\ \hline pTSN-Bisa-Mm-GapC & 1000 & 0.025 & 5.3 & 0.07 & 0.07 \\ pSSN12 Didi-MrBBS-IspA & 1000 & 0.025 & 3.6 & 0.05 & 0.05 \\ pdCas9-sgRNA-GapA & & & & & \\ \hline \end{tabular}  
* Initial yield and productivity are calculated based on values in the early cultivation period (0–46 h).  
\end{table} 
Table 1: Summary of fed-batch fermentations of (\(-\))-\(\alpha\)-bisabololol by engineered _E. coli_. were purchased from Promega (Madison, WI, USA) and oligonucleotide synthesis were conducted by Bioneer (Daejeon, Korea). The _E. coli_ codon-optimized _mvaK1_ gene of _M. mazei_ (GenBank accession number: KKI06753.1) was synthesized by Bioneer (Figure S1). The synthesized _mvaK1_ was PCR-amplified with MM-IF and MM-IR primers, and the plasmid backbone was amplified with the MM-VF and MM-VR primers from pSNA-MrBBS-IspA. The two PCR-amplicons were assembled via the Gibson Assembly Method [30] using Gibson Assembly Master Mix (NEB), resulting in the construction of the pTSN-Bisa-Mm plasmid. The _mvaK1_ from _S. aureus_ was amplified with SA-IF and SA-IR primers from pTM-BBS, and the plasmid for the backbone was obtained from pSNA-MrBBS-IspA by PCR with a set of primers of SA-VF and SA-VR, followed by assembly with the Gibson assembly method. The resulting plasmid was named pTSN-Bisa-Sa. For the construction of pSEVA231-Bisa-Mm, the first fragment containing the MVA pathway gene, _ispA_ and _MrBBS_ were amplified in pTSN-Bisa-Mm using primers of pBBR1-IF and pBBR1-IR. The second fragment harboring the kanamycin-resistant gene and pBBR1 origin was amplified from pSEVA231 as a template using pBBR1-VF and pBBR1-VR primers. The fragments were assembled via Gibson Assembly method. To construct pSSN12 Didi-MrBBS-IspA, the _MrBBS_ and _ispA_ including _trc_ promoter gene were amplified using Didi-I-F and Didi-I-R primers from the pTSN-Bisa-Mm plasmid. The vector backbone containing genes encoding enzymes of the lower MVA pathway was amplified using Didi-V-F and Didi-V-R primers from the pSSN12 Didi plasmid. Two amplified fragments were then assembled via the Gibson Assembly kit. The _E. coli_ codon-optimized _gapC_ gene from _C.