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

ABSTRACT: (-)-?-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. (-)-?-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 (-)-?-bisabolol plays a key role in the development of its sustainable production from renewable feedstock.Here, we created an Escherichia coli strain producing (-)-?-bisabolol at high titer and developed an in situ extraction method of (-)-?-bisabolol, using natural vegetable oils. We expressed a recently identified (-)-?-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 (-)-?-bisabolol precursor farnesyl diphosphate. Combinatorial expression of the exogenous mevalonate pathway and farnesyl diphosphate synthase enabled a dramatic increase in (-)-?-bisabolol production in the shake flask culture (80 mg/L) and 5 L bioreactor culture (342 mg/L) of engineered E. coli harboring (-)-?-bisabolol synthase. Fed-batch fermentation using a 50 L fermenter was conducted after optimizing culture conditions, resulting in efficient (-)-?-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 (-)-?-bisabolol during fermentation and showed high yield recovery (>98%).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 (-)-?-bisabolol at large scale.

SUBMITTER: Han GH

PROVIDER: S-EPMC5101696 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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

Han Gui Hwan GH Kim Seong Keun SK Yoon Paul Kyung-Seok PK Kang Younghwan Y Kim Byoung Su BS Fu Yaoyao Y Sung Bong Hyun BH Jung Heung Chae HC Lee Dae-Hee DH Kim Seon-Won SW Lee Seung-Goo SG

Microbial cell factories 20161108 1

<h4>Background</h4>(-)-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 ro ...[more]

PMID: 27825357

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Project description:BackgroundVanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details.ResultsEffect of plasmid copy number in metabolic engineering of E. coli for the synthesis of vanillin has been evaluated by the use of genes encoding feruloyl-CoA synthetase and feruloyl hydratase/aldolase from Pseudomonas fluorescens BF13. The higher vanillin production yield was obtained using resting cells of E. coli strain JM109 harbouring a low-copy number vector and a promoter exhibiting a low activity to drive the expression of the catabolic genes. Optimization of the bioconversion of ferulic acid to vanillin was accomplished by a response surface methodology. The experimental conditions that allowed us to obtain high values for response functions were 3.3 mM ferulic acid and 4.5 g/L of biomass, with a yield of 70.6% and specific productivity of 5.9 micromoles/g x min after 3 hours of incubation. The final concentration of vanillin in the medium was increased up to 3.5 mM after a 6-hour incubation by sequential spiking of 1.1 mM ferulic acid. The resting cells could be reused up to four times maintaining the production yield levels over 50%, thus increasing three times the vanillin obtained per gram of biomass.ConclusionFerulic acid can be efficiently converted to vanillin, without accumulation of undesirable vanillin reduction/oxidation products, using E. coli JM109 cells expressing genes from the ferulic acid-degrader Pseudomonas fluorescens BF13. Optimization of culture conditions and bioconversion parameters, together with the reuse of the biomass, leaded to a final production of 2.52 g of vanillin per liter of culture, which is the highest found in the literature for recombinant strains and the highest achieved so far applying such strains under resting cells conditions.

Dataset Information

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

Publications

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

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