title: Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme.
abstract: BACKGROUND
  Plant terpenoids are known for their diversity, stereochemical complexity, and their commercial interest as pharmaceuticals, food additives, and cosmetics. Developing biotechnology approaches for the production of these compounds in heterologous hosts can increase their market availability, reduce their cost, and provide sustainable production platforms. In this context, we aimed at producing the antimicrobial diterpenoid isopimaric acid from Sitka spruce. Isopimaric acid is synthesized using geranylgeranyl diphosphate as a precursor molecule that is cyclized by a diterpene synthase in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4.
  
  
  RESULTS
  We transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co-expression resulted in 3-fold increase in the accumulation of both isopimaradiene and isopimaric acid detected using GC-MS and LC-MS methodology. We also showed that modifying or deleting the transmembrane helix of CYP720B4 does not alter the enzyme activity and led to successful accumulation of isopimaric acid in the infiltrated leaves. Furthermore, we demonstrated that a modified membrane anchor is a prerequisite for a functional CYP720B4 enzyme when the chloroplast targeting peptide is added. We report the accumulation of 45-55 μg/g plant dry weight of isopimaric acid four days after the infiltration with the modified enzymes.
  
  
  CONCLUSIONS
  It is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer to photosynthesis paves the way for light-driven biosynthesis of valuable terpenoids.
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, UNIK Center for Synthetic Biology, Villum Research Center "Plant Plasticity", University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Thiyagarajan
authors.initials: T
authors.lastname: Gnanasekaran
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, UNIK Center for Synthetic Biology, Villum Research Center "Plant Plasticity", University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Vavitsas
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, UNIK Center for Synthetic Biology, Villum Research Center "Plant Plasticity", University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.|Present address: Plant and Microbial Biology, University of California, 371 Koshland Hall, Berkeley, CA 94720 USA.
authors.forename: Johan
authors.initials: J
authors.lastname: Andersen-Ranberg
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, UNIK Center for Synthetic Biology, Villum Research Center "Plant Plasticity", University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Agnieszka Zygadlo
authors.initials: AZ
authors.lastname: Nielsen
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, UNIK Center for Synthetic Biology, Villum Research Center "Plant Plasticity", University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Carl Erik
authors.initials: CE
authors.lastname: Olsen
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, UNIK Center for Synthetic Biology, Villum Research Center "Plant Plasticity", University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Björn
authors.initials: B
authors.lastname: Hamberger
authors.affiliation: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, UNIK Center for Synthetic Biology, Villum Research Center "Plant Plasticity", University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: Poul Erik
authors.initials: PE
authors.lastname: Jensen
keywords: Chloroplasts; Cytochrome P450; Diterpenoids; Isopimaric acid; Protein engineering