Abstract:

Objective Pregnenolone serves as a key precursor for synthesizing various steroid hormones, holding significant industrial applications. However, its traditional chemical synthesis process is complex and environmentally burdensome. Constructing a pregnenolone biosynthesis system using Saccharomyces cerevisiae as a chassis provides a new technological pathway for its green and efficient production. Method The recombinant strain CY03 of S. cerevisiae, previously constructed in our laboratory for high-yield campesterol production, was taken as the research object. P450scc systems from different sources were introduced into S. cerevisiae to compare their catalytic performance, and the optimal enzyme combination was screened out. The multi-copy sites of S. cerevisiae rDNA were further utilized to achieve multi-copy integration of the P450scc system, thereby enhancing its expression. Concurrently, we attempted to enhance the electron transport efficiency between CYP87A4 and its redox partner ATR1 using protein fusion strategies. We also optimized key factors in the culture medium, including carbon source, nitrogen source, and inorganic salts, to enhance pregnenolone synthesis. Result The combination of CYP87A4 from Digitalis lanata and ATR1 from Arabidopsis thaliana exhibited the best catalytic performance, achieving a pregnenolone titer of 3.75 mg/L. After multi-copy integration of the P450scc system at the rDNA locus, a recombinant strain with a copy number of 5 was successfully screened, which increased the pregnenolone production to 14.51 mg/L, which was 3.87 times higher than that of the single-copy strain. The protein fusion strategy failed to further enhance the efficiency of pregnenolone synthesis. Through optimization of the culture medium conditions, the pregnenolone production was further increased to 16.82 mg/L under the following conditions: 20 g/L glucose, 10 g/L yeast extract, 20 g/L peptone, and 0.3 g/L K⁺. Conclusion We successfully constructed and optimized a biosynthetic system for de novo pregnenolone synthesis in S. cerevisiae. By screening P450scc system sources, performing multi-copy integration, and optimizing fermentation conditions, we significantly enhanced the yield. This study provides a feasible engineering strategy and an experimental basis for the green microbial manufacturing of steroid compounds.

Key words: Saccharomyces cerevisiae, pregnenolone, de novo synthesis, culture medium optimization