(−)-α-Bisabolol is produced from depyrophosphorylation of the prenyl diphosphate precursor FPP. Therefore, synthesis of FPP from IPP and DMAPP by FPP synthase, encoded by the ispA gene, plays a key role in the improved production of (−)-α-bisabolol. Furthermore, high concentrations of prenyl diphosphates, such as FPP, are toxic to E. coli cell growth . FPPs needs to be efficiently converted into downstream products by TPSs to reduce its toxicity. To increase the synthesis of the cellular terpenoid biosynthesis intermediate FPP, and (−)-α-bisabolol production, overexpression of an FPP synthase, encoded by the ispA gene, was performed. The ispA gene of E. coli was inserted downstream of the MrBBS gene in the plasmid pSNA-MrBBS, resulting in the pSNA-MrBBS-IspA plasmid (Fig. 1b). This plasmid was transformed into E. coli DH5α and cultivated in TB medium containing 1% (w/v) glycerol at 30 °C for 48 h using a two-phase culture without the addition of IPTG. Overexpression of the ispA gene improved (−)-α-bisabolol production compared with pSNA-MrBBS alone. After 48 h, a concentration of 79.7 mg/L of (−)-α-bisabolol was obtained in a culture of DH5α with pSNA-MrBBS-IspA. This was twofold higher than the 39.7 mg/L of (−)-α-bisabolol produced by DH5α with pSNA-MrBBS alone (Fig. 6a), without significant difference in cell growth (Additional file 1: Figure S3D). This suggested that sufficient supply of FPP is important to improve (−)-α-bisabolol production in E. coli. While overexpressing the ispA gene, 0.05 mM IPTG at OD600 ~0.5 was added to induce expression of the MVA pathway and MrBBS genes. Similar to the results without ispA overexpression, (−)-α-bisabolol production decreased from 87.8 mg/L (no IPTG) to 40 mg/L (−)-α-bisabolol (0.05 mM IPTG, Fig. 6b) with no significant difference in cell growth (Additional file 1: Figure S3D). To monitor the time-course of (−)-α-bisabolol production, batch fermentation of E. coli DH5α harboring the pSNA-MrBBS-IspA plasmid, using a 1 L fermenter, was conducted at 30 °C in the absence of IPTG for 93 h (Fig. 6c). (−)-α-bisabolol production of 214 mg/L was achieved and cell growth reached an OD600 value of 11.5 after 93 h. MVA and acetate were the main byproducts, although the acetate was consumed after glycerol depletion at 48 h. The solubility of heterologous enzymes increases at lower growth temperatures because the transcription rate is slower, leading to better protein folding in E. coli [37, 38]. In addition, improved solubility of metabolic pathway enzymes at low temperature enhances the production yield . Therefore, we performed the batch fermentation at 25 °C under the same conditions. Indeed, (−)-α-bisabolol production was 342 mg/L after fermentation for 93 h, approximately 1.6-fold higher than that obtained at 30 °C, and cell growth decreased by 25% (OD600 8.7, Fig. 6d).Fig. 6IspA overexpression and batch fermentation of engineered E. coli. a Improvement of (−)-α-bisabolol production in E. coli DH5α transformed with pSNA-MrBBS-IspA compared with DH5α harboring the pSNA-MrBBS plasmid. Cells were cultivated in TB medium containing 10 g/L glycerol at 30 °C for 48 h using two-phase culture without the addition of IPTG. Data represent averages from three replicate cultures; error bars show SD, b effects of IPTG addition on (−)-α-bisabolol production in E. coli DH5α harboring pSNA-MrBBS-IspA. Culture conditions were the same as in Fig. 6a, except for the addition of IPTG. IPTG was added to each culture to a final concentration of 0.05 mM when the OD600 reached 0.5. Data represent averages from three replicate cultures; error bars show SD, c time profile of batch fermentation of E. coli DH5α with pSNA-MrBBS-IspA in TB medium containing 10 g/L of glycerol as carbon source at 30 °C, n-dodecane was overlaid up to 20% (v/v) of culture volume to continuously extract (−)-α-bisabolol from the culture broth during fermentation. d Time profile of batch fermentation of E. coli DH5α with pSNA-MrBBS-IspA in TB medium containing 10 g/L of glycerol as carbon source at 25 °C. n-Dodecane was overlaid up to 20% (v/v) of culture volume to continuously extract (−)-α-bisabolol from the culture broth during fermentation