通过酿酒酵母的组合优化高效生产 22(R)-羟基胆固醇。

Efficient production of 22(R)-hydroxycholesterol via combination optimization of Saccharomyces cerevisiae.

机构信息

Key Laboratory of Medical Molecule Science and Pharmaceutical Engineering, Ministry of Industry and Information Technology, Institute of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing, China.

School of Life Science, Beijing Institute of Technology, Beijing, China.

出版信息

Biotechnol J. 2024 Jul;19(7):e2400286. doi: 10.1002/biot.202400286.

DOI:10.1002/biot.202400286

PMID:39014927

Abstract

22(R)-hydroxycholesterol (22(R)-HCHO) is a crucial precursor of steroids biosynthesis with various biological functions. However, the production of 22(R)-HCHO is expensive and unsustainable due to chemical synthesis and extraction from plants or animals. This study aimed to construct a microbial cell factory to efficiently produce 22(R)-HCHO through systems metabolic engineering. First, we tested 7-dehydrocholesterol reductase (Dhcr7s) and cholesterol C22-hydroxylases from different sources in Saccharomyces cerevisiae, and the titer of 22(R)-HCHO reached 128.30 mg L in the engineered strain expressing Dhcr7 from Columba livia (ClDhcr7) and cholesterol 22-hydroxylase from Veratrum californicum (VcCyp90b27). Subsequently, the 22(R)-HCHO titer was significantly increased to 427.78 mg L by optimizing the critical genes involved in 22(R)-HCHO biosynthesis. Furthermore, hybrid diploids were constructed to balance cell growth and 22(R)-HCHO production and to improve stress tolerance. Finally, the engineered strain produced 2.03 g L of 22(R)-HCHO in a 5-L fermenter, representing the highest 22(R)-HCHO titer reported to date in engineered microbial cell factories. The results of this study provide a foundation for further applications of 22(R)-HCHO in various industrially valuable steroids.

摘要

22(R)-羟基胆固醇（22(R)-HCHO）是类固醇生物合成的关键前体，具有多种生物学功能。然而，由于化学合成和从植物或动物中提取，22(R)-HCHO 的生产既昂贵又不可持续。本研究旨在通过系统代谢工程构建微生物细胞工厂来高效生产 22(R)-HCHO。首先，我们在酿酒酵母中测试了来自不同来源的 7-脱氢胆固醇还原酶（Dhcr7s）和胆固醇 C22-羟化酶，在表达来自鸽（ClDhcr7）的 Dhcr7 和来自 Veratrum californicum（VcCyp90b27）的胆固醇 22-羟化酶的工程菌株中，22(R)-HCHO 的产量达到 128.30 mg/L。随后，通过优化参与 22(R)-HCHO 生物合成的关键基因，将 22(R)-HCHO 的产量显著提高到 427.78 mg/L。此外，构建了杂种二倍体以平衡细胞生长和 22(R)-HCHO 生产，并提高了应激耐受性。最后，该工程菌株在 5-L 发酵罐中生产了 2.03 g/L 的 22(R)-HCHO，这是迄今为止在工程微生物细胞工厂中报道的最高 22(R)-HCHO 产量。本研究结果为进一步将 22(R)-HCHO 应用于各种工业价值的类固醇提供了基础。