Pregnenolone is an important steroid precursor with broad pharmaceutical applications. However, its traditional production mainly relies on semi-synthesis from plant-derived diosgenin, which is associated with resource limitations and high production costs. In this study, a pregnenolone-producing Saccharomyces cerevisiae strain was systematically engineered by optimizing intracellular iron utilization, heme biosynthesis, and NADPH regeneration to improve the intracellular environment supporting the plant-derived P450scc system (CYP87A4). First, engineering of the iron homeostasis network revealed that overexpression of AFT2 combined with deletion of CCC1 increased pregnenolone production to 18.87 mg/L, representing a 30.05% improvement over the parental strain. Subsequently, enhancement of heme biosynthesis through HEM13 overexpression and SHM1 deletion increased intracellular heme content by 72.70% and further improved pregnenolone production to 25.00 mg/L. To strengthen the reducing power supply, several NADPH-generating enzymes were evaluated. Co-overexpression of POS5 and ZWF1 proved to be the most effective strategy, increasing intracellular NADPH content and the NADPH/NADP⁺ ratio by 70.98% and 105.00% respectively, and elevating pregnenolone production to 32.58 mg/L. Finally, fermentation conditions were optimized through single-factor experiments and an L9(34) orthogonal design. Under the optimal conditions of 20 g/L glucose, 40 g/L mixed nitrogen source, 1% inoculum size, 24 h seed cultivation time, and 0.3 g/L K⁺ supplementation, the pregnenolone titer reached 48.66 mg/L, representing a 49.36% increase compared with that before process optimization. These results demonstrate that coordinated engineering of iron metabolism, heme biosynthesis, and NADPH regeneration can improve intracellular conditions associated with CYP87A4 function and provide a promising strategy for improving microbial production of steroid precursors.
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Funding
the National Natural Science Foundation of China(No. 22578169)
the Fundamental Research Funds for the Central Universities(Grant JUSRP202504023)
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