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    Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica

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    InteMethod Referecne corrected.pdf (846.9Kb)
    Supplementary materials - v2.pdf (296.6Kb)
    Links to Files
    https://pubs.acs.org/doi/10.1021/acssynbio.8b00535
    Permanent Link
    https://doi.org/10.1021/acssynbio.8b00535
    http://hdl.handle.net/11603/12855
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    • UMBC Chemical, Biochemical & Environmental Engineering Department
    • UMBC Faculty Collection
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    Author/Creator
    Lv, Yongkun
    Edwards, Harley
    Zhou, Jingwen
    Xu, Peng
    Date
    2019-01-29
    Type of Work
    9 pages
    Text
    journal articles postprints
    Citation of Original Publication
    Yongkun Lv, Harley Edwards, Jingwen Zhou, and Peng Xu, 1.Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica, ACS Synthetic Biology Article ASAP, 2019, DOI: 10.1021/acssynbio.8b00535
    Rights
    This item is likely protected under Title 17 of the U.S. Copyright Law. Unless on a Creative Commons license, for uses protected by Copyright Law, contact the copyright holder or the author.
    This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Synth. Biol., Article ASAP, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://pubs.acs.org/doi/10.1021/acssynbio.8b00535
    Access to this item will begin on January 29, 2020
    Subjects
    chromosome integration
    Cre-loxP
    26s rDNA
    Y. lipolytica
    flavonoids
    cytochrome c P450 enzymes
    Abstract
    Conventional plasmid-based gene expression tends to introduce genetic instability and gene copy number variations that lead to degenerated production. The limited number of auxotrophic markers in Yarrowia lipolytica also restricts our ability to perform iterative genetic modifications and manipulate long gene clusters. To overcome these limitations, we combined the high recombination efficiency of the Cre-loxP system and the high integration rate of 26s rDNA, and developed a versatile framework to iteratively integrate multicopy metabolic pathways in Y. lipolytica. We demonstrated the efficient genome integration of a plant-derived flavonoid pathway at random sites with multiple copies. Transient expression of Cre recombinase enabled efficient marker removal and allowed for the next round of genome integration. Investigating the recombination events demonstrated that the iterative integration is happening at sufficiently high rates (more than 80%) without disrupting the previous integration. Both the flavonoid precursor pathway and the plant-derived cytochrome c P450 enzymes were functionally integrated to improve flavonoid and hydroxylated flavonoid production. The engineered strains produced 71.2 mg/L naringenin, 54.2 mg/L eriodyctiol, and 48.1 mg/L taxifolin. The reported work provides a versatile platform to iteratively integrate functional gene clusters at high copy numbers. This work may streamline and expand our capability to build efficient microbial cell factories for high-value natural products and commodity chemical production in Y. lipolytica.


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    Albin O. Kuhn Library & Gallery
    University of Maryland, Baltimore County
    1000 Hilltop Circle
    Baltimore, MD 21250
    www.umbc.edu/scholarworks

    Contact information:
    Email: scholarworks-group@umbc.edu
    Phone: 410-455-3544


    If you wish to submit a copyright complaint or withdrawal request, please email mdsoar-help@umd.edu.