Secretory production of 7-dehydrocholesterol by engineered Saccharomyces cerevisiae

Background: 7-Dehydrocholesterol (7-DHC) can be directly converted to vitamin D3 by UV irradiation and de novo synthesis of 7-DHC in engineered Saccharomyces cerevisiae has been recognized as an attractive substitution to traditional chemical synthesis. Introduction of sterol extracellular transport pathway for the secretory production of 7-DHC is a promising approach to achieve higher titer and simplify the downstream purification processing.Methods and results: A series of genes involved in ergosterol pathway were combined reinforced and reengineered in S. cerevisiae. A biphasic fermentation system was introduced and 7-DHC was found to be enriched in oil-phase with an increased titer by 1.5-folds. Quantitative PCR revealed that say1, atf2, pdr5, pry1-3 involved in sterol storage and transport were all significantly induced in sterol overproduced strain. To enhance the secretion capacity, lipid transporters of pathogen-related yeast proteins (Pry), Niemann-Pick disease type C2 (NPC2), ATP-binding cassette (ABC)-family, and their homologues were screened. Both individual and synergetic overexpression of Plant pathogenesis Related protein-1 (Pr-1) and Sterol transport1 (St1) largely increased the de novo biosynthesis and secretory productivity of 7-DHC, and the final titer reached 28.2 mg g(-1) with a secretion ratio of 41.4%, which was 26.5-folds higher than the original strain. In addition, the cooperation between Pr-1 and St1 in sterol transport was further confirmed by confocal microscopy, molecular docking, and directed site-mutation.Conclusion: Selective secretion of different sterol intermediates was characterized in sterol over-produced strain and the extracellular export of 7-DHC developed in present study significantly improved the cell biosynthetic capacity, which offered a novel modification idea for 7-DHC de novo biosynthesis by S. cerevisiae cell factory.

Automated summary

This study improved the biosynthetic and secretory productivity of 7-Dehydrocholesterol (7-DHC) in Saccharomyces cerevisiae by reinforcing and reengineering the ergosterol pathway genes, introducing a biphasic fermentation system, and overexpressing lipid transporters. The selective secretion of different sterol intermediates was characterized, and the cooperation between Pr-1 and St1 in sterol transport was confirmed. The improved cell biosynthetic capacity offers a novel modification approach for 7-DHC de novo biosynthesis.