Coupling yeast golden gate and VEGAS for efficient assembly of the violacein pathway in saccharomyces cerevisiae

James Chuang, Jef D. Boeke, Leslie A. Mitchell

    Research output: Chapter in Book/Report/Conference proceedingChapter

    Abstract

    The ability to express non-native pathways in genetically tractable model systems is important for fields such as synthetic biology, genetics, and metabolic engineering. Here we describe a modular and hierarchical strategy to assemble multigene pathways for expression in S. cerevisiae. First, discrete promoter, coding sequence, and terminator parts are assembled in vitro into Transcription Units (TUs) flanked by adapter sequences using “yeast Golden Gate” (yGG), a type IIS restriction enzyme-dependent cloning strategy. Next, harnessing the natural capacity of S. cerevisiae for homologous recombination, TUs are assembled into pathways and expressed using the “Versatile Genetic Assembly System” (VEGAS) in yeast. Coupling transcription units constructed by yGG with VEGAS assembly is a generic and flexible workflow to achieve pathway expression in S. cerevisiae. This protocol describes assembly of a five TU pathway for yeast production of violacein, a pigment derived from Chromobacterium violaceum.

    Original languageEnglish (US)
    Title of host publicationMethods in Molecular Biology
    PublisherHumana Press Inc.
    Pages211-225
    Number of pages15
    DOIs
    StatePublished - 2018

    Publication series

    NameMethods in Molecular Biology
    Volume1671
    ISSN (Print)1064-3745

    Keywords

    • Hierarchical DNA assembly
    • Metabolic engineering
    • Saccharomyces cerevisiae
    • Versatile Genetic Assembly System (VEGAS)
    • Violacein
    • Yeast Golden Gate (yGG)

    ASJC Scopus subject areas

    • Molecular Biology
    • Genetics

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