n-Dodecane has been mostly used for the extraction of terpenoids produced in microorganisms due to its relatively low volatility, enabling continuous extraction over multiple days . The (−)-α-bisabolol produced in flask culture and batch fermentation could be efficiently extracted from the broth to the overlaid n-dodecane phase. The in situ recovery of (−)-α-bisabolol from the n-dodecane phase during culture can reduce production costs by simplifying both the harvest and purification processes. However, n-dodecane is not an economically viable extracting solvent. We assessed various natural and inexpensive vegetable oils for selective (−)-α-bisabolol extraction from fermentation broth. As shown in Table 2, all tested vegetable oils showed extraction yields ranging from 96.6 to 98.8%, which are comparable to that of n-dodecane (99.5%). Among them, canola oil (98.8%) was the best extracting oil and was used for the extraction of (−)-α-bisabolol during fed-batch fermentation. A greener extraction of (−)-α-bisabolol plays a key role in the application of (−)-α-bisabolol produced from fermentation broth to pharmaceutical and cosmetic products. We successfully used vegetable oils as natural, cost-effective, and biodegradable extractors for in situ extraction of (−)-α-bisabolol during fermentation. Moreover, due to the difference in polarity between the fermentation broth (water based) and vegetable oils, there is a phase separation, resulting in isolation of oil from the broth containing cell debris. This in situ (−)-α-bisabolol separation from the fermentation broth can also alleviate production inhibition and improve (−)-α-bisabolol production, which will be of interest in industrial processes. To examine the production performance of (−)-α-bisabolol under conditions more relevant to industrial processes, we conducted (−)-α-bisabolol fermentation in a 50 L fermenter with a fed-batch mode. E. coli DH5α, containing pSNA-MrBBS-IspA, was grown at 25 °C with 10% (v/v) canola oil as an extracting solution. The final (−)-α-bisabolol titer reached 3.3 g/L after 90 h of fermentation, which was almost 8.4-fold higher than that obtained from the batch fermentation at 25 °C (Fig. 7). At the beginning of fermentation, MVA was accumulated until depletion of the initial 10 g/L glycerol. After complete depletion of initial glycerol, MVA was consumed and (−)-α-bisabolol accumulated continuously up to 9.1 g/L at the end of fermentation (Fig. 7). The specific (−)-α-bisabolol production and productivity reached 0.18 g/g dry cell weight (DCW) and 1.24 g/L/day, respectively. A previous study reported the production of 8 mg/L of (−)-α-bisabolol from S. cerevisiae by overexpressing the MrBBS gene for 4 days . In this study, two-phase fed-batch fermentation of the DH5α strain expressing the MrBBS, entire MVA pathway, and ispA genes yielded 9.1 g/L of (−)-α-bisabolol after 150 h, a 1137-fold increase from that reported in the previous study. Since the (−)-α-bisabolol intermediate, mevalonate, accumulated after glycerol depletion, (−)-α-bisabolol production would increase if we increase expression of the genes of the lower MVA pathway. Such adjustments will be the content of further work to improve (−)-α-bisabolol production.Table 2Recovery yield of (−)-α-bisabolol using common vegetable oilsExtracting solutionExtraction yield (%)Soybean oil98.5Canola oil98.8Corn oil98.5Sunflower oil96.7 n-Dodecane99.5 Fig. 7Fed-batch fermentation of E. coli DH5α in TB medium supplemented with 10 g/L of glycerol as initial carbon source. After complete depletion of glycerol, glycerol was fed at 3 g/L/h. Concentrations of acetate, mevalonate, and glycerol were determined by HPLC and (−)-α-bisabolol content was measured using GC. Canola oil 10% (v/v) instead of n-dodecane 20% (v/v) was used to overlay 30 L TB medium in a 50 L fermenter