Multi-copy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica
Date
2008
Authors
Maharajh D.
Roth R.
Lalloo R.
Simpson C.
Mitra R.
Gorgens J.
Ramchuran S.
Journal Title
Journal ISSN
Volume Title
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Abstract
Epoxide hydrolases (EHs) of fungal origin have the ability to catalyze the enantioselective hydrolysis of epoxides to their corresponding diols. However, wild type fungal EHs are limited in substrate range and enantioselectivity. Additionally, the production of fungal epoxide hydrolase (EH) by wild-type strains is typically very low. In the present study, the EH-encoding gene from Rhodotorula araucariae was functionally expressed in Yarrowia lipolytica, under the control of a growth phase inducible hp4d promoter, in a multi-copy expression cassette. The transformation experiments yielded a positive transformant, with a final EH activity of 220 U/g dw in shake-flask cultures. Evaluation of this transformant in batch fermentations resulted in ∼ 7-fold improvement in EH activity over the flask scale. Different constant specific feed rates were tested in fed-batch fermentations, resulting in an EH activity of 1,750 U/g dw at a specific feed rate of ∼ 0.1 g/g/h, in comparison to enzyme production levels of 0.3 U/g dw for the wild type R. araucariae and 52 U/g dw for an Escherichia coli recombinant strain expressing the same gene. The expression of EH in Y. lipolytica using a multi-copy cassette demonstrates potential for commercial application. © 2008 Springer-Verlag.
Description
Keywords
Cell culture, Enantioselectivity, Enzyme activity, Fermentation, Hydrolysis, Enzyme production, Epoxide hydrolases, Fed-batch fermentation, Rhodotorula araucariae, Yarrowia lipolytica, Fungi, epoxide, epoxide hydrolase, enzyme activity, gene expression, hydrolysis, yeast, article, bacterial strain, enantioselectivity, enzyme activity, enzyme mechanism, Escherichia coli, fed batch fermentation, fungal gene, fungal strain, fungus, gene cassette, gene expression, hp4d gene, hydrolysis, nonhuman, nucleotide sequence, promoter region, Rhodotorula araucariae, wild type, Yarrowia lipolytica, Bioreactors, Biotechnology, Epoxide Hydrolases, Epoxy Compounds, Fermentation, Fungal Proteins, Gene Expression, Recombinant Fusion Proteins, Rhodotorula, Yarrowia, Escherichia coli, Rhodotorula araucariae, Yarrowia lipolytica
Citation
Applied Microbiology and Biotechnology
79
2
79
2