title: In vivo assembly of DNA-fragments in the moss, Physcomitrella patens.
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.
authors.affiliation: Copenhagen Plant Science Centre, VILLUM Research Center for Plant Plasticity, Center for Synthetic Biology "bioSYNergy", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
authors.forename: Brian Christopher
authors.initials: BC
authors.lastname: King
authors.affiliation: Copenhagen Plant Science Centre, VILLUM Research Center for Plant Plasticity, Center for Synthetic Biology "bioSYNergy", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Vavitsas
authors.affiliation: Copenhagen Plant Science Centre, VILLUM Research Center for Plant Plasticity, Center for Synthetic Biology "bioSYNergy", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.|Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.|Department of Systems Biology, Technical University of Denmark, Søltofts Plads, 2800 Kgs. Lyngby, Denmark.
authors.forename: Nur Kusaira Binti Khairul
authors.initials: NK
authors.lastname: Ikram
authors.affiliation: Copenhagen Plant Science Centre, VILLUM Research Center for Plant Plasticity, Center for Synthetic Biology "bioSYNergy", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
authors.forename: Josephine
authors.initials: J
authors.lastname: Schrøder
authors.affiliation: Copenhagen Plant Science Centre, VILLUM Research Center for Plant Plasticity, Center for Synthetic Biology "bioSYNergy", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
authors.forename: Lars B
authors.initials: LB
authors.lastname: Scharff
authors.forename: Jean-Étienne
authors.initials: JÉ
authors.lastname: Bassard
authors.affiliation: Copenhagen Plant Science Centre, VILLUM Research Center for Plant Plasticity, Center for Synthetic Biology "bioSYNergy", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
authors.forename: Björn
authors.initials: B
authors.lastname: Hamberger
authors.affiliation: Copenhagen Plant Science Centre, VILLUM Research Center for Plant Plasticity, Center for Synthetic Biology "bioSYNergy", Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
authors.forename: Poul Erik
authors.initials: PE
authors.lastname: Jensen
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Søltofts Plads, 2800 Kgs. Lyngby, Denmark.
authors.forename: Henrik Toft
authors.initials: HT
authors.lastname: Simonsen
mesh_terms: D019068:Bryopsida / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D000072669:Gene Editing; D055786:Gene Knockout Techniques; D059765:Homologous Recombination; D011401:Promoter Regions, Genetic
chemical_list: D018744:DNA, Plant