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.

Effect of an exponential feeding regime on the production ofRhodotorula araucariaeepoxide hydrolase inYarrowia lipolytica

“…The inoculum for all experiments was prepared by inoculating a single cryovial of Y. lipolytica (Maharajh et al. 2008) into 700 ml of growth medium (1·5% w/v yeast extract, 0·89% w/v malt extract, 0·67% w/v glucose) contained in a 1800‐ml Fernback flask.…”

“…2008). Constant glucose feeding in fed‐batch culture increased EH production substantially in comparison to batch culture, with final production levels being two orders of magnitude higher than in wild type R. araucariae (Maharajh et al. 2008).…”

“…This coupled with the commercial importance of EH has led to its expression in various recombinant hosts, including Yarrowia lipolytica (Visser et al. 2002; Labuschagne and Albertyn 2007; Maharajh et al. 2008).…”

“…2008). The EH gene from Rhodotorula araucariae was previously expressed in Y. lipolytica using a multi‐copy vector (Maharajh et al. 2008).…”

Genetic modification of the marine‐derived yeast Yarrowia lipolytica with high‐protein content using a GPI‐anchor‐fusion expression system

“…The digestion rate of single cell protein of yeast cells is generally above 80%. 2 In general, yeast cells can grow very quickly, the highest dry cell weight concentration during batch fermentation can reach 14.8 g/L, 4 and the fermentation period is very short. 5 Compared with algal cultivation, it is very easy to manage the large-scale yeast cell production in the fermentor.…”