Rate-limiting steps in the Saccharomyces cerevisiae ergosterol pathway: towards improved ergosta-5,7-dien-3β-ol accumulation by metabolic engineering.

Ergosterol is the predominant nature sterol constituent of plasma membrane in Saccharomyces cerevisiae. Herein, the biosynthetic pathway of ergosterol was proposed to be metabolically engineered for the efficient production of ergosta-5,7-dien-3β-ol, which is the precursor of vitamin D4. By target disruption of erg5, involved in the end-steps of post-squalene formation, predominantly accumulated ergosta-5,7-dien-3β-ol (4.12 mg/g dry cell weight). Moreover, the rate-limiting enzymes of ergosta-5,7-dien-3β-ol biosynthesis were characterized. Overexpression of Hmg1p led to a significant accumulation of squalene, and induction of Erg1p/Erg11p expression raised the yield of both total sterols and ergosta-5,7-dien-3β-ol with no obvious changes in growth behavior. Furthermore, the transcription factor allele upc2-1 was overexpressed to explore the effect of combined induction of rate-limiting enzymes. Compared with an obviously enhanced yield of ergosterol in the wild-type strain, decreases of both the ergosta-5,7-dienol levels and the total sterol yield were found in Δerg5-upc2-1, probably due to the unbalanced NADH/NAD+ ratio observed in the erg5 knockouts, suggesting the whole-cell redox homeostasis was also vital for end-product biosynthesis. The data obtained in this study can be used as reference values for the production of sterol-related intermediates involved in the post-squalene biosynthetic pathway in food-grade S. cerevisiae strains.