Plants produce more than 10.000 diterpenoid compounds of which the large majority is involved in specialized metabolism, while a few are involved in general metabolism. Specialized metabolism diterpenoids have functions in interactions of plants with other organisms and selected ones are utilized by humanity in biopharmaceuticals or as industrial bioproducts. Yields and purity of diterpenoids purified from natural sources or made by chemical synthesis are generally insufficient for large-volume or high-end applications, thus alternative sources are needed. Synthetic biology, where heterologous pathways have been reconstructed in host production organisms is an attractive lternative, which holds the promise to enable a scalable, costeffective and table supply of natural products. Knowledge about the genes and mechanisms nvolved in the original pathway is a prerequisite for such heterologous production.

Since only small changes in the amino acid sequence can influence the roduct outcome of a diterpene synthase (diTPS), prediction of the catalytic activity diTPS of a is not possible purely based on phylogenetic relationship. Thus, functional characterization is required in to determine the catalytic activity of an unknown diTPS. Today, popular hosts for functional genomics and pathway reconstitution are bacterial and fungal systems. However, increasing xperimental evidence has indicated that the expression host can influence the observed products of such assays. For functional expression and aracterization of diTPSs deriving from the plant kingdom, a plant expression host offers several advantages such as the presence of all relevant compartments (plastids and endoplasmic reticulum) and the universal C5 building blocks for isoprenoid biosynthesis. In addition, a plant based xpression host is compatible with native codon usage, and through the conserved mechanisms of protein targeting and posttranslational odifications, has the capacity to produce functional enzymes. To further explore plant based expression and characterization of diterpenoid pathway genes, two different plant expression hosts were chosen: stable P. patens expression and transient N. benthamiana expression.

The presented data highlights that P. patens has a simple chemical background and a great capacity of producing endogenous as well as non-endogenous diterpenoids. High rates of homologous recombination were exploited to generate viable P. patens lines with disrupted PpCPS/KS unctionality. These kaurenoid free Ppcps/ks lines were utilized to express the bifunctional diTPS PaLAS from Norway spruce, for the first time in planta (Paper 2). By direct comparison to assays with PaLAS expressed in microbial systems it could be shown that P. patens provided conditions that resulted in unique in planta PaLAS products, confirming that the product profile of a diTPS can be influenced by the expression host and assay conditions. Thus, the P. patens based expression platform, developed during this study, represents a molecular tool complementing and extending existing nowledge about diterpene synthase activity obtained from functional characterization using microbial based in vitro and in vivo strategies.

This study represents the first attempt of using kaurenoid free P. patens lines to redirect GGPP into production of non-endogenous diterpene products. However, based on the limited number of P. patens lines generated in this study, and the large variations in expression levels, it was not possible to conclude if kaurenoid free P. patens lines have a higher production capacity for non-endogenous diterpenoids, compared to wild type P. patens.

This study also represents the first evidence of using transient co-expression in N. benthamiana of functional diTPSs and a P450 enzyme. It was shown, that this rapid and flexible expression system facilitated large scale coupled and non-coupled diTPS in vivo assays using diTPS genes from several plant pecies using the native codons and plastid targeting sequences. This indicated that the transient N. benthamiana expression constitutes an attractive platform for the rapid discovery and functional characterization of diterpenoid pathways, complementing the most recent next-generation sequence driven pathway discovery approaches (Paper 4).

For molecular biology, laboratories interested in establishing these two plant expression platforms, the practical experiences made during these studies have been made available in a special isoprenoid issue of “Methods in Molecular Biology” (Paper 1 and 3), but have been shared with a number of labs around the globe already. The content of this thesis will enable esearchers to explore the benefits of these plant expression hosts for characterization of terpenoid genes and contribute to a better understanding of terpenoid biosynthesis in planta.