Fermentative production and direct extraction of (−)-α-bisabolol in metabolically engineered Escherichia coli

Abstract

Background(−)-α-Bisabolol, also known as levomenol, is an unsaturated sesquiterpene alcohol that has mainly been used in pharmaceutical and cosmetic products due to its anti-inflammatory and skin-soothing properties. (−)-α-Bisabolol is currently manufactured mainly by steam-distillation of the essential oils extracted from the Brazilian candeia tree that is under threat because its natural habitat is constantly shrinking. Therefore, microbial production of (−)-α-bisabolol plays a key role in the development of its sustainable production from renewable feedstock.ResultsHere, we created an Escherichia coli strain producing (−)-α-bisabolol at high titer and developed an in situ extraction method of (−)-α-bisabolol, using natural vegetable oils. We expressed a recently identified (−)-α-bisabolol synthase isolated from German chamomile (Matricaria recutita) (titer: 3 mg/L), converted the acetyl-CoA to mevalonate, using the biosynthetic mevalonate pathway (12.8 mg/L), and overexpressed farnesyl diphosphate synthase to efficiently supply the (−)-α-bisabolol precursor farnesyl diphosphate. Combinatorial expression of the exogenous mevalonate pathway and farnesyl diphosphate synthase enabled a dramatic increase in (−)-α-bisabolol production in the shake flask culture (80 mg/L) and 5 L bioreactor culture (342 mg/L) of engineered E. coli harboring (−)-α-bisabolol synthase. Fed-batch fermentation using a 50 L fermenter was conducted after optimizing culture conditions, resulting in efficient (−)-α-bisabolol production with a titer of 9.1 g/L. Moreover, a green, downstream extraction process using vegetable oils was developed for in situ extraction of (−)-α-bisabolol during fermentation and showed high yield recovery (>98%).ConclusionsThe engineered E. coli strains and economically viable extraction process developed in this study will serve as promising platforms for further development of microbial production of (−)-α-bisabolol at large scale.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0588-2) contains supplementary material, which is available to authorized users.

Efficient production of Pseudoionone with multipathway engineering in Escherichia coli

“…For example, increasing idi copies enhanced (–)‐α‐bisabolol production in E. coli (Han et al . ), replacing the native promoter of the MEP pathway genes (including dxs ) with the strong bacteriophage T5 promoter resulted in the production of β‐carotene at high titres (6 mg g −1 dry cell weight) (Yuan et al . ).…”

Overexpression of key enzymes of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway for improving squalene production in Escherichia coli

“…IspA is also considered to be a key enzyme for terpenoids biosynthesis (Han et al, 2016), however ispA is not used extensively with MEP pathway genes but is widely used in the MVA pathway. Combination of dxs, idi and ispA can increase squalene production in YSS12 by up to 71-fold when compared with the strain that only harbors two copies of SQS.…”

“…DXS and IDI have been reported as the keyenzymes in the MEP pathway for increasing terpenoid production in E. coli (Yuan et al, 2006; and squalene production (Ghimire et al, 2009). Overexpression of genes dxr (Lv et al, 2016), ispDF Yuan et al, 2006), ispG (Liu et al, 2014) and ispA (Han et al, 2016) were able to enhance terpenoids production. On the contrary, other studies have shown that overexpression of dxr coupled with dxs produced a similar isoprene level when compared with the dxs overproduction strain (Xue and Ahring, 2011), and overexpression of ispDF together with dxs and idi resulted in decrease in terpenoids production (Zhou et al, 2012).…”

Techno‐economic assessment of the use of solvents in the scale‐up of microbial sesquiterpene production for fuels and fine chemicals

“…Alternatively, this separation step could be bypassed by choosing solvents compatible with the final product formulation. Examples include the use of methyl oleate or isopropyl myristate in the production of amorphadiene, canola oil in the production of the sesquiterpene alcohol bisabolol, and farnesene as solvent in the production of the monoterpene limonene …”

“…As a result, the product is not a homogeneous continuous phase but a stable emulsion. Although the formation of sesquiterpene emulsions is usually not mentioned in laboratory‐scale studies, this problem has been reported at larger scales . Reported recovery methods include inducing phase inversion and obtaining an emulsion of water in a continuous oil phase (w/o), which is separated afterwards by centrifugation.…”