Bach, Søren Spanner5; King, Brian Christopher6; Zhan, Xin5; Simonsen, Henrik Toft5; Hamberger, Björn Robert5
1 Section for Plant Biochemistry, Department of Plant and Environmental Sciences, Faculty of Science, Københavns Universitet2 Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, Faculty of Science, Københavns Universitet3 Plant Biochemistry, Department of Plant Biology, Faculty of Life Sciences, Københavns Universitet4 Section for Molecular Plant Biology, Department of Plant and Environmental Sciences, Faculty of Science, Københavns Universitet5 Plant Biochemistry, Department of Plant Biology, Faculty of Life Sciences, Københavns Universitet6 Section for Molecular Plant Biology, Department of Plant and Environmental Sciences, Faculty of Science, Københavns Universitet
Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure, such as streamlining of large scale Agrobacterium infiltration and upregulation of the upstream pathways, transient in planta heterologous expression quickly reaches limitations when used for production of terpenoids. Stable integration of transgenes into the nuclear genome of the moss Physcomitrella patens has already been widely recognized as a viable alternative for industrial-scale production of biopharmaceuticals. For expression of terpenoid biosynthetic genes, and reconstruction of heterologous pathways, Physcomitrella has unique attributes that makes it a very promising biotechnological host. These features include a high native tolerance to terpenoids, a simple endogenous terpenoid profile, convenient genome editing using homologous recombination, and cultivation techniques that allow up-scaling from single cells in microtiter plates to industrial photo-bioreactors. Beyond its use for functional characterization of terpenoid biosynthetic genes, engineered Physcomitrella can be a green biotechnological platform for production of terpenoids. Here, we describe two complementary and simple procedures for stable nuclear transformation of Physcomitrella with terpenoid biosynthetic genes, selection and cultivation of transgenic lines, and metabolite analysis of terpenoids produced in transgenic moss lines. We also provide tools for metabolic engineering through genome editing using homologous recombination.
Methods in Molecular Biology: Methods and Protocols, 2014, p. 257-271