High-level diterpene production by transient expression in Nicotiana benthamiana

Abstract

BackgroundCharacterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.ResultsAgrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2.ConclusionWith this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.

A Tandem Array of ent-Kaurene Synthases in Maize with Roles in Gibberellin and More Specialized Metabolism

“…ZmTPS1, ZmKSL5, and ZmKSL3 Targeting and in Planta KS Activity Nicotiana benthamiana has been reported to be a good host for plant metabolic engineering, particularly including diterpene production, with transient expression of the relevant TPSs (Brückner and Tissier, 2013). In an initial trial, transient expression of ZmTPS1, ZmKSL3, or ZmKSL5 under control of the 35S promoter in N. benthamiana did not lead to the detection of any new terpenes by GC-MS analysis.…”

“…Transient overexpression of ZmTPS1, ZmKSL3, and ZmKSL5 in N. benthamiana was carried out as described by Brückner and Tissier (2013). Briefly, Agrobacterium tumefaciens strain GV3101 was transformed with pCAMBIA 2300-eGFP constructs, with or without stop codons, and the resulting recombinant strains infiltrated into 5-week-old N. benthamiana, either with or without An2, as well as P19 to suppress posttranscriptional silencing (Voinnet et al, 2003).…”

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize

“…To test for the activity of ZmKSL4 in specialized diterpenoid metabolism, we utilized the combinatorial functionality of class II and class I diTPSs of different plant species, which can be exploited to probe diTPS functions (Brückner and Tissier 2013;Zerbe et al, 2013;Kitaoka et al, 2015). An established Escherichia coli coexpression system (Morrone et al, 2010) was used to analyze the coupled activities of ZmKSL4 with (1) the ent-CPP synthase ZmAN2, (2) the rice syn-CPP synthase (OsCPS4; Xu et al, 2004), and (3) the grand fir (Abies grandis) abietadiene synthase variant D621A that produces CPP of normal [i.e.…”

“…Modular enzyme networks largely determine the variations of diterpene profiles in closely and distantly related plant species (Peters, 2010;Hamberger and Bak, 2013;Zerbe and Bohlmann, 2015). Naturally occurring modularity in diterpenoid biosynthesis further enables the engineering of combinatorial diTPS and P450 expression systems in microbial and plant host systems to accelerate the discovery of terpenoid pathways (Brückner and Tissier, 2013;Lange and Ahkami, 2013;Kitaoka et al, 2015;Zerbe and Bohlmann, 2015).…”

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.…”