A High-Throughput Workflow for CRISPR/Cas9 Mediated Combinatorial Promoter Replacements and Phenotype Characterization in Yeast

    Research output: Contribution to journalArticleResearchpeer-review

    • 2 Citations

    Abstract

    Due to the rapidly increasing sequence information on gene variants generated by evolution and the improved abilities to engineer novel biological activities, microbial cells can be evolved for the production of a growing spectrum of compounds. For high productivity, efficient carbon channeling toward the end product is a key element. In large scale production systems the genetic modifications that ensure optimal performance cannot be dependent on plasmid-based regulators, but need to be engineered stably into the host genome. Here, a CRISPR/Cas9 mediated high-throughput workflow for combinatorial and multiplexed replacement of native promoters with synthetic promoters and the following high-throughput phenotype characterization in the yeast Saccharomyces cerevisiae is described. The workflow is demonstrated with three central metabolic genes, ZWF1, PGI1, and TKL1 encoding a glucose-6-phosphate dehydrogenase, phosphoglucose isomerase, and transketolase, respectively. The synthetic promoter donor DNA libraries are generated by PCR and transformed to yeast cells. A 50% efficiency is achieved for simultaneous replacement at three individual loci using short 60-bp flanking homology sequences in the donor promoters. Phenotypic strain characterization is validated and demonstrated using liquid handling automation and 150μL cultivation volume in 96-well plate format. The established workflow offers a robust platform for automated engineering and improvement of yeast strains.

    LanguageEnglish
    Article number1700593
    JournalBiotechnology Journal
    Volume13
    Issue number9
    DOIs
    Publication statusPublished - 1 Sep 2018

    Fingerprint

    Clustered Regularly Interspaced Short Palindromic Repeats
    Workflow
    Yeasts
    Phenotype
    Transketolase
    Glucose-6-Phosphate Isomerase
    Glucosephosphate Dehydrogenase
    Automation
    Sequence Homology
    Gene Library
    Genes
    Saccharomyces cerevisiae
    Plasmids
    Carbon
    Genome
    Polymerase Chain Reaction

    Keywords

    • Automation
    • CRISPR
    • High-throughput
    • Metabolic engineering
    • Saccharomyces cerevisiae
    • Yeast

    OKM Publication Types

    • A1 Refereed journal article

    OKM Open Access Status

    • 0 Not Open Access

    ASJC Scopus subject areas

    • Applied Microbiology and Biotechnology
    • Molecular Medicine

    Cite this

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    title = "A High-Throughput Workflow for CRISPR/Cas9 Mediated Combinatorial Promoter Replacements and Phenotype Characterization in Yeast",
    abstract = "Due to the rapidly increasing sequence information on gene variants generated by evolution and the improved abilities to engineer novel biological activities, microbial cells can be evolved for the production of a growing spectrum of compounds. For high productivity, efficient carbon channeling toward the end product is a key element. In large scale production systems the genetic modifications that ensure optimal performance cannot be dependent on plasmid-based regulators, but need to be engineered stably into the host genome. Here, a CRISPR/Cas9 mediated high-throughput workflow for combinatorial and multiplexed replacement of native promoters with synthetic promoters and the following high-throughput phenotype characterization in the yeast Saccharomyces cerevisiae is described. The workflow is demonstrated with three central metabolic genes, ZWF1, PGI1, and TKL1 encoding a glucose-6-phosphate dehydrogenase, phosphoglucose isomerase, and transketolase, respectively. The synthetic promoter donor DNA libraries are generated by PCR and transformed to yeast cells. A 50{\%} efficiency is achieved for simultaneous replacement at three individual loci using short 60-bp flanking homology sequences in the donor promoters. Phenotypic strain characterization is validated and demonstrated using liquid handling automation and 150μL cultivation volume in 96-well plate format. The established workflow offers a robust platform for automated engineering and improvement of yeast strains.",
    keywords = "Automation, CRISPR, High-throughput, Metabolic engineering, Saccharomyces cerevisiae, Yeast",
    author = "Joosu Kuivanen and Sami Holmstr{\"o}m and Birgitta Lehtinen and Merja Penttil{\"a} and Jussi J{\"a}ntti",
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    A High-Throughput Workflow for CRISPR/Cas9 Mediated Combinatorial Promoter Replacements and Phenotype Characterization in Yeast. / Kuivanen, Joosu; Holmström, Sami; Lehtinen, Birgitta; Penttilä, Merja; Jäntti, Jussi.

    In: Biotechnology Journal, Vol. 13, No. 9, 1700593, 01.09.2018.

    Research output: Contribution to journalArticleResearchpeer-review

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    AU - Kuivanen, Joosu

    AU - Holmström, Sami

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    AU - Penttilä, Merja

    AU - Jäntti, Jussi

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