State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
Appl Biochem Biotechnol. 2021 Aug;193(8):2443-2454. doi: 10.1007/s12010-021-03540-w. Epub 2021 Mar 13.
In the present work, we tried to identify the mechanism why by which the steroid alcohols accumulated when hydroxypropyl-β-cyclodextrin (HP-β-CD) was present to enhance the sterol conversion rate. Compared with the bioconversion system without HP-β-CD, the reaction rate was greatly improved in presence of HP-β-CD, but the steroid alcohols largely accumulated concurrently. In a reaction system with an enhanced reaction rate, the higher intracellular NADH/NAD level was detected, and the production of steroid alcohols increased also. Mycobacterium neoaurum mutants with higher KshA activity (3-ketosteroid 9α-hydrolase, a monooxygenase hydroxylating the nucleus at C-9 at the expense of NAD(P)H consumption) reduced the steroid alcohol production, and in the meantime, the NADH/NAD level was decreased consequently. Further research found that oxygen availability was seriously inhibited by the cyclodextrin in a reaction system. These results indicated that NADH formed in the bioconversion was not properly regenerated via the respiratory chain because of the poor oxygen bioavailability. The inhibitory effect of cyclodextrin on oxygen bioavailability is a key factor for the metabolic flux redistribution toward steroid alcohols in phytosterol resting cells bioconversion.
在本工作中，我们试图确定在羟丙基-β-环糊精（HP-β-CD）存在时促进固醇转化率的机制。与没有 HP-β-CD 的生物转化系统相比，在 HP-β-CD 的存在下，反应速率大大提高，但同时固醇醇也大量积累。在反应速率提高的反应系统中，检测到更高的细胞内 NADH/NAD 水平，固醇醇的产量也增加。具有更高 KshA 活性（3-酮类固醇 9α-水解酶，一种单加氧酶，以 NAD(P)H 消耗为代价在核的 C-9 处羟基化）的分枝杆菌突变体减少了固醇醇的产生，同时 NADH/NAD 水平也随之降低。进一步的研究发现，在反应系统中环糊精严重抑制了氧气的可用性。这些结果表明，由于氧气生物利用度差，生物转化过程中形成的 NADH 无法通过呼吸链得到适当的再生。环糊精对氧气生物利用度的抑制作用是植物固醇休止细胞生物转化中代谢通量向固醇醇重新分配的关键因素。
