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 Pry (Pathogen-related yeast proteins), NPC2(Niemann-Pick disease type C2), ABC(ATP-binding cassette)-family and their homologues were screened. Both individual and synergetic overexpression of Pr-1 (Plant pathogenesis Related protein-1) and St1 (Sterol transport1) largely increased the de novo biosynthesis and secretory productivity of 7-DHC, and the final titer reached 28.2 mg/g 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. This article is protected by copyright. All rights reserved.
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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