Secretory production of 7‐dehydrocholesterol by engineered Saccharomyces cerevisiae

Abstract

AbstractBackground7‐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 resultsA 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.ConclusionSelective 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.