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.
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Acknowledgement
The authors would like to thank Mr Khama Mathiba for his assistance with the GC analysis and INRA for supplying the genetic tools and markers as well as Dr Michel Labuschagne of UOFS for his assistance with real time PCR and information supplied. This work was funded by the Innovation Fund in South Africa, and the authors express the utmost gratitude for this funding.
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Maharajh, D., Roth, R., Lalloo, R. et al. Multi-copy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica . Appl Microbiol Biotechnol 79, 235–244 (2008). https://doi.org/10.1007/s00253-008-1420-7
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DOI: https://doi.org/10.1007/s00253-008-1420-7