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.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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