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

Abstract

Abstract22(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−1 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−1 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−1 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.