Heterologous Stable Expression of Terpenoid Biosynthetic Genes Using the Moss Physcomitrella patens

Abstract

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.

Can mosses serve as model organisms for forest research?

“…To date, several human proteins are produced in moss, e.g., vascular endothelial growth factor (Baur et al 2005), erythropoietin (Weise et al 2007), and human complement factor H (Buettner-Mainik et al 2011). In addition, moss is used as production platform for secondary metabolites such as terpenoids (Bach et al 2014). …”

Additional diterpenes from Physcomitrella patens synthesized by copalyl diphosphate/kaurene synthase ( Pp CPS/KS)

“…patens (Gransden ecotype, International Moss Stock Center #40001) was grown as previously described [14] in a growth chamber with a 16 hour light/8 hour dark cycle at 25°C and with light intensity at Wm-2 (700-800 µEinstein just above the plant).…”

“…polyethylene glycol (PEG) mediated transformation and stable lines were recovered and analyzed by GC-MS as previously described [14,16]. Genomic DNA from stable P. patens lines without detectible ent-kaurene was prepared as previously described [14].…”

“…Genomic DNA from stable P. patens lines without detectible ent-kaurene was prepared as previously described [14]. Sequencing of PCR products using the primers 5'-AAGCCGACTTCAACATGTG-3' (oSSB94), 5'-AACCCGAAGCTCTTCCAC-3' (oSSB95), 5'-CCTGTGCAAGGTAAGAAGATGG-3' (oCL77) and 5'-CTCTGTCTCTCCCACAATCCTCC-3' (oCL252)…”

Enzymes for Synthetic Biology of Ambroxide-Related Diterpenoid Fragrance Compounds

“…Building on the experience gained with established metabolic engineering platforms for high-value terpenoids, such as the anticancer diterpenoid drug Taxol and the sesquiterpenoid artemisinin used in the treatment of malaria [24,26,63], new microbial and plant-based platforms for the production of natural and nonnatural diterpene bioproducts are currently being developed in several laboratories [17,26,[64][65][66]. In the following sections, we describe three examples of diterpene biosynthesis, sclareol production in clary sage, cis-abienol formation in balsam fir, and DRA biosynthesis in the Pinaceae, to highlight recent research on the genetic and enzymatic diversity of diterpenoid metabolism and its application for the fragrance industry.…”