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 bio-synthetic pathway of ergosterol was proposed to be metabolically engineered for the efficient production of ergosta-5,7-dien3 beta-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-3 beta-ol (4.12 mg/g dry cell weight). Moreover, the rate-limiting enzymes of ergosta-5,7-dien-3 beta-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-3 beta-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 wildtype strain, decreases of both the ergosta-5,7-dienol levels and the total sterol yield were found in Delta erg5-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.