Maharajh Dheepak, Roth Robyn, Lalloo Rajesh, Simpson Clinton, Mitra Robin, Görgens Johann, Ramchuran Santosh
CSIR Biosciences, Private Bag X2, Modderfontein 1645, South Africa.
Appl Microbiol Biotechnol. 2008 May;79(2):235-44. doi: 10.1007/s00253-008-1420-7.
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.
真菌来源的环氧化物水解酶（EHs）能够催化环氧化物对映选择性水解生成相应的二醇。然而，野生型真菌EHs的底物范围和对映选择性有限。此外，野生型菌株产生真菌环氧化物水解酶（EH）的量通常非常低。在本研究中，来自南洋红酵母的EH编码基因在多拷贝表达盒中，在生长阶段诱导型hp4d启动子的控制下，在解脂耶氏酵母中实现了功能性表达。转化实验产生了一个阳性转化体，在摇瓶培养中最终EH活性为220 U/g干重。在分批发酵中对该转化体进行评估，结果表明EH活性比摇瓶规模提高了约7倍。在补料分批发酵中测试了不同的恒定比进料速率，在约0.1 g/g/h的比进料速率下，EH活性达到1750 U/g干重，相比之下，野生型南洋红酵母的酶产量水平为0.3 U/g干重，表达相同基因的大肠杆菌重组菌株的酶产量水平为52 U/g干重。使用多拷贝盒在解脂耶氏酵母中表达EH显示出商业应用潜力。
