Cloning, multicopy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in yarrowia lipolytica

Abstract

Epoxide hydrolases (EHs) of fungal origin have the ability to catalyse the enantioselective hydrolysis of epoxides to their corresponding diols. However wild type fungal EHs are limited in the substrate range and enantioselectivity, additionally wild type fungal EH productivities are relatively low. Recombinant DNA technology has been previously used to overproduce these enzymes in expression systems such as E. coli and A.niger and P. pastoris. EH encoding genes from Rhodotorula araucariae were cloned and functionally expressed in Y. lipolytica, under the control of a growth inducible hp4d promoter. The transformation experiments yielded only two positive multicopy transformants, which were assessed in flask cultures. The selected transformant demonstrated a 4 fold enhanced EH activity over the transformant. The transformant was then evaluated in batch and fed batch fermentations, where the batch fermentations resulted in - 50% improved EH activity from flask evaluations. In fed batch fermentations, different specific feed rates were tested. A specific feed rate of 0.1 g.g-1.h-1 resulted in the highest EH activity of 1750 mU.mg dw-1, compared to maximum production levels of 0.3 mU.mg dw-1 for the wild type R. araucariae and 52 mU.mg dw-1 E. coli. A 2.7-fold increase was observed from shake-flask fermentation to the fed-batch fermentation