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


RESULTS
Here, 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%).


CONCLUSIONS
The 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.
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.
Gui Hwan
GH
Han
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.|Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon, 34113, Republic of Korea.
Seong Keun
SK
Kim
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.|Department of Chemical and Biological Engineering, Korea University, Seoul, 02841, Republic of Korea.
Paul Kyung-Seok
PK
Yoon
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.
Younghwan
Y
Kang
Department of Biotechnology, Chonnam National University, Yeosu, 550749, Republic of Korea.
Byoung Su
BS
Kim
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.
Yaoyao
Y
Fu
Bioenergy and Biochemical Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.
Bong Hyun
BH
Sung
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea.
Heung Chae
HC
Jung
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea. dhlee@kribb.re.kr.|Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon, 34113, Republic of Korea. dhlee@kribb.re.kr.
Dae-Hee
DH
Lee
Division of Applied Life Science (BK21 Plus), PMBBRC, Gyeongsang National University, Jinju, 52828, Republic of Korea. swkim@gnu.ac.kr.
Seon-Won
SW
Kim
Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Republic of Korea. sglee@kribb.re.kr.|Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon, 34113, Republic of Korea. sglee@kribb.re.kr.
Seung-Goo
SG
Lee
(−)-α-Bisabolol; (−)-α-Bisabolol synthase; Escherichia coli; Farnesyl diphosphate synthase; In situ extraction; Mevalonate pathway; Vegetable oils