In vivo assembly of DNA-fragments in the moss, Physcomitrella patens

Brian Christopher King, Konstantinos Vavitsas, Nur Kusaira Binti Khairul Ikram, Josephine Schroder, Lars B. Scharff, Bjorn Hamberger, Poul Erik Jensen, Henrik Toft Simonsen

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    Abstract

    Direct assembly of multiple linear DNA fragments via homologous recombination, a phenomenon known as in vivo assembly or transformation associated recombination, is used in biotechnology to assemble DNA constructs ranging in size from a few kilobases to full synthetic microbial genomes. It has also enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments in P. patens with three examples of effective genome editing: we (i) efficiently deleted a genomic locus for diterpenoid metabolism yielding a biosynthetic knockout, (ii) introduced a salt inducible promoter, and (iii) re-routed endogenous metabolism into the formation of amorphadiene, a precursor of high-value therapeutics. These proof-of-principle experiments pave the way for more complex and increasingly flexible approaches for large-scale metabolic engineering in plant biotechnology.
    Original languageEnglish
    Article number25030
    JournalScientific Reports
    Volume6
    Number of pages7
    ISSN2045-2322
    DOIs
    Publication statusPublished - 2016

    Bibliographical note

    This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

    Keywords

    • Genetic engineering
    • Plant biotechnology

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