School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, China.
School of Life Sciences, Shanghai University, Shanghai 200444, China.
Biomolecules. 2021 Nov 17;11(11):1710. doi: 10.3390/biom11111710.
24-Methylene-cholesterol is a necessary substrate for the biosynthesis of physalin and withanolide, which show promising anticancer activities. It is difficult and costly to prepare 24-methylene-cholesterol via total chemical synthesis. In this study, we engineered the biosynthesis of 24-methylene-cholesterol in by disrupting the two enzymes (i.e., ERG4 and ERG5) in the yeast's native ergosterol pathway, with ERG5 being replaced with the DHCR7 (7-dehydrocholesterol reductase) enzyme. Three versions of DHCR7 originating from different organisms-including the DHCR7 from (PhDHCR7) newly discovered in this study, as well as the previously reported OsDHCR7 from and XlDHCR7 from -were assessed for their ability to produce 24-methylene-cholesterol. XlDHCR7 showed the best performance, producing 178 mg/L of 24-methylene-cholesterol via flask-shake cultivation. The yield could be increased up to 225 mg/L, when one additional copy of the expression cassette was integrated into the yeast genome. The 24-methylene-cholesterol-producing strain obtained in this study could serve as a platform for characterizing the downstream enzymes involved in the biosynthesis of physalin or withanolide, given that 24-methylene-cholesterol is a common precursor of these chemicals.
24-亚甲基胆固醇是法呢醇和醉茄内酯生物合成的必要底物，具有有前景的抗癌活性。通过全化学合成制备 24-亚甲基胆固醇既困难又昂贵。在这项研究中，我们通过破坏酵母中天然麦角固醇途径中的两种酶（即 ERG4 和 ERG5），并用 DHCR7（7-去氢胆固醇还原酶）酶取代 ERG5，在 中工程化 24-亚甲基胆固醇的生物合成。评估了来自三个不同生物体的三种 DHCR7 版本（包括本研究中新发现的 PhDHCR7，以及之前报道的 OsDHCR7 来自 和 XlDHCR7 来自 ），以评估它们生产 24-亚甲基胆固醇的能力。XlDHCR7 表现出最佳性能，通过摇瓶培养生产 178mg/L 的 24-亚甲基胆固醇。当将 表达盒的一个额外拷贝整合到酵母基因组中时，产量可增加到 225mg/L。鉴于 24-亚甲基胆固醇是这些化学物质的共同前体，本研究获得的 24-亚甲基胆固醇生产菌株可以作为表征参与法呢醇或醉茄内酯生物合成的下游酶的平台。
