title: Multi-copy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica.
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 approximately 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 approximately 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.
authors.affiliation: CSIR Biosciences, Private Bag X2, Modderfontein 1645, South Africa. DMaharajh@csir.co.za
authors.forename: Dheepak
authors.initials: D
authors.lastname: Maharajh
authors.forename: Robyn
authors.initials: R
authors.lastname: Roth
authors.forename: Rajesh
authors.initials: R
authors.lastname: Lalloo
authors.forename: Clinton
authors.initials: C
authors.lastname: Simpson
authors.forename: Robin
authors.initials: R
authors.lastname: Mitra
authors.forename: Johann
authors.initials: J
authors.lastname: Görgens
authors.forename: Santosh
authors.initials: S
authors.lastname: Ramchuran
mesh_terms: D019149:Bioreactors; D001709:Biotechnology; D004851:Epoxide Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004852:Epoxy Compounds / Q000378:metabolism*; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis; D015870:Gene Expression; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012248:Rhodotorula / Q000201:enzymology / Q000235:genetics*; D025062:Yarrowia / Q000235:genetics*
chemical_list: D004852:Epoxy Compounds; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D004851:Epoxide Hydrolases