To implement the de novo production of (−)-α-bisabolol in E. coli DH5α, the gene of MrBBS, encoding the (−)-α-bisabolol synthase, was codon-optimized for E. coli and introduced via the pTSN-MrBBS plasmid (Fig. 1b). The resulting transformant cells were cultured in LB medium overlaid with 20% (v/v) n-dodecane for 48 h. Two-phase cultures have been successfully used to extract toxic, water-immiscible or volatile products [24–26] and n-dodecane was successfully used for extraction of (−)-α-bisabolol from the culture broth of S. cerevisiae . As expected, in the present study, (−)-α-bisabolol was extracted into the n-dodecane phase in the two-phase E. coli culture. As the (−)-α-bisabolol was recovered in n-dodecane without significant residual amounts left in the cells (Additional file 1: Figure S2), (−)-α-bisabolol purification in a later step will be facilitated. The n-dodecane phase was used for GC analysis to measure (−)-α-bisabolol concentrations. There was a peak at 25.6 min in the n-dodecane phase sample of E. coli DH5α with pTSN-MrBBS, corresponding to the standard (−)-α-bisabolol compound dissolved in n-dodecane (Fig. 3a). Mass spectrometry confirmed that the peak at 25.6 min was (−)-α-bisabolol (Fig. 3b, c) and a maximum concentration of 3 mg/L (−)-α-bisabolol was produced by the endogenous MEP pathway and exogenous MrBBS enzyme in E. coli DH5α. The peak was not observed in two-phase cultures of DH5α cells containing an empty plasmid as a control experiment (Fig. 3a). The (−)-α-bisabolol synthase gene, MrBBS, used in this study was recently isolated from German chamomile and, unlike other TSPs, exclusively synthesizes (−)-α-bisabolol as a single terpene product . Several TPSs producing α-bisabolol as a single major product have been previously cloned and biochemically characterized. However, these enzymes usually produce undesirable isomers with different structures or α-bisabolol of unknown stereochemistry [27–29]. The MrBBS enzyme has shown unique catalytic features including the formation of a single enantiopure (−)-α-bisabolol indicating the possibility of a biotechnological application to natural (−)-α-bisabolol production in S. cerevisiae . Here, we have expressed the codon-optimized synthetic MrBBS in E. coli and successfully produced (−)-α-bisabolol (titer: 3 mg/L). A similar result has been observed when attempting to express other plant terpene synthases in E. coli [30, 31].Fig. 3GC and GC–MS analysis of n-dodecane extracts from E. coli expressing the MrBBS gene. a Total ion chromatograms of an authentic (−)-α-bisabolol standard (100 mg/L) and the n-dodecane extracts from the E. coli expressing MrBBS (pTSN-MrBBS) or containing an empty vector (pTrc99A). b Mass spectra of (−)-α-bisabolol standard. c Mass spectra of (−)-α-bisabolol produced in E. coli cells harboring the plasmid pTSN-MrBBS. Each cell was cultivated in LB medium overlaid with 20% (v/v) n-dodecane at 30 °C for 48 h