鉴定和工程化参与分枝杆菌甾醇分解代谢初始步骤的胆固醇氧化酶。

Identification and engineering of cholesterol oxidases involved in the initial step of sterols catabolism in Mycobacterium neoaurum.

机构信息

State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.

出版信息

Metab Eng. 2013 Jan;15:75-87. doi: 10.1016/j.ymben.2012.10.005. Epub 2012 Nov 17.

DOI:10.1016/j.ymben.2012.10.005

PMID:23164577

Abstract

Mycobacteria have been modified to transform sterols to produce valuable steroids. Here, we demonstrated that the oxidation of sterols to sterones is a rate-limiting step in the catabolic pathway of sterols in Mycobacterium neoaurum. Two cholesterol oxidases ChoM1 and ChoM2 involved in the step were identified in M. neoaurum and the ChoM2 shared up to 45% identity with other cholesterol oxidases. We demonstrated that the combination of ChoM1 and ChoM2 plays a significant role in this step. Accordingly, we developed a strategy to overcome this rate-limiting step by augmenting the activity of cholesterol oxidases in M. neoaurum strains to enhance their transformation productivity of sterols to valuable steroids. Our results indicated that the augmentation of ChoM2 achieved 5.57g/l androst-1,4-diene-3,17-dione in M. neoaurum NwIB-01MS and 6.85g/l androst-4-ene-3,17-dione in M. neoaurum NwIB-R10, greatly higher than the original yield, 3.87g/l androst-1,4-diene-3,17-dione and 4.53g/l androst-4-ene-3,17-dione, respectively.

摘要

分枝杆菌已被改造为将甾醇转化为有价值的甾体。在这里，我们证明甾醇到甾酮的氧化是分枝杆菌中新金色单胞菌甾醇分解代谢途径中的限速步骤。鉴定出两种参与该步骤的胆固醇氧化酶 ChoM1 和 ChoM2，并在新金色单胞菌中发现 ChoM2 与其他胆固醇氧化酶的同一性高达 45%。我们证明 ChoM1 和 ChoM2 的组合在该步骤中起着重要作用。因此，我们开发了一种通过增强新金色单胞菌菌株中胆固醇氧化酶的活性来克服这一限速步骤的策略，以提高它们将甾醇转化为有价值的甾体的转化生产力。我们的结果表明，ChoM2 的增强可使新金色单胞菌 NwIB-01MS 中的雄甾-1,4-二烯-3,17-二酮达到 5.57g/l，而在新金色单胞菌 NwIB-R10 中的雄甾-4-烯-3,17-二酮达到 6.85g/l，大大高于原始产量，分别为 3.87g/l 雄甾-1,4-二烯-3,17-二酮和 4.53g/l 雄甾-4-烯-3,17-二酮。